Inbred corn plant 3323 and seeds thereof

ABSTRACT

According to the invention, there is provided an inbred corn plant designated 3323. This invention thus relates to the plants, seeds and tissue cultures of the inbred corn plant 3323, and to methods for producing a corn plant produced by crossing the inbred corn plant 3323 with itself or with another corn plant, such as another inbred. This invention further relates to corn seeds and plants produced by crossing the inbred plant 3323 with another corn plant, such as another inbred, and to crosses with related species. This invention further relates to the inbred and hybrid genetic complements of the inbred corn plant 3323, and also to the SSR and genetic isozyme typing profiles of inbred corn plant 3323.

BACKGROUND OF THE INVENTION

[0001] This application claims the priority of U.S. Provisionalapplication Ser. No. 60/179,374, filed Jan. 31, 2000, the disclosure ofwhich is specifically incorporated herein by reference in its entirety.

1. FIELD OF THE INVENTION

[0002] The present invention relates generally to the field of cornbreeding. In particular, the invention relates to inbred corn seed andplants designated 3323, and derivatives and tissue cultures thereof

2. DESCRIPTION OF RELATED ART

[0003] The goal of field crop breeding is to combine various desirabletraits in a single variety/hybrid. Such desirable traits include greateryield, better stalks, better roots, resistance to insecticides,herbicides, pests, and disease, tolerance to heat and drought, reducedtime to crop maturity, better agronomic quality, higher nutritionalvalue, and uniformity in germination times, stand establishment, growthrate, maturity, and fruit size.

[0004] Breeding techniques take advantage of a plant's method ofpollination. There are two general methods of pollination: a plantself-pollinates if pollen from one flower is transferred to the same oranother flower of the same plant. A plant cross-pollinates if pollencomes to it from a flower on a different plant.

[0005] Corn plants (Zea mays L.) can be bred by both self-pollinationand cross-pollination. Both types of pollination involve the cornplant's flowers. Corn has separate male and female flowers on the sameplant, located on the tassel and the ear, respectively. Naturalpollination occurs in corn when wind blows pollen from the tassels tothe silks that protrude from the tops of the ear shoot.

[0006] Plants that have been self-pollinated and selected for type overmany generations become homozygous at almost all gene loci and produce auniform population of true breeding progeny, a homozygous plant. A crossbetween two such homozygous plants produces a uniform population ofhybrid plants that are heterozygous for many gene loci. Conversely, across of two plants each heterozygous at a number of loci produces apopulation of hybrid plants that differ genetically and are not uniform.The resulting non-uniformity makes performance unpredictable.

[0007] The development of uniform corn plant hybrids requires thedevelopment of homozygous inbred plants, the crossing of these inbredplants, and the evaluation of the crosses. Pedigree breeding andrecurrent selection are examples of breeding methods used to developinbred plants from breeding populations. Those breeding methods combinethe genetic backgrounds from two or more inbred plants or various otherbroad-based sources into breeding pools from which new inbred plants aredeveloped by selfing and selection of desired phenotypes. The newinbreds are crossed with other inbred plants and the hybrids from thesecrosses are evaluated to determine which of those have commercialpotential.

[0008] The pedigree breeding method involves crossing two genotypes.Each genotype can have one or more desirable characteristics lacking inthe other; or, each genotype can complement the other. If the twooriginal parental genotypes do not provide all of the desiredcharacteristics, other genotypes can be included in the breedingpopulation. Superior plants that are the products of these crosses areselfed and selected in successive generations. Each succeedinggeneration becomes more homogeneous as a result of self-pollination andselection. Typically, this method of breeding involves five or moregenerations of selfing and selection: S₁→S₂; S₂→S₃; S₃→S₄; S₄→S₅, etc.After at least five generations, the inbred plant is consideredgenetically pure.

[0009] Backcrossing can also be used to improve an inbred plant.Backcrossing transfers a specific desirable trait from one inbred ornon-inbred source to an inbred that lacks that trait. This can beaccomplished, for example, by first crossing a superior inbred (A)(recurrent parent) to a donor inbred (non-recurrent parent), whichcarries the appropriate locus or loci for the trait in question. Theprogeny of this cross are then mated back to the superior recurrentparent (A) followed by selection in the resultant progeny for thedesired trait to be transferred from the non-recurrent parent. Afterfive or more backcross generations with selection for the desired trait,the progeny are heterozygous for loci controlling the characteristicbeing transferred, but are like the superior parent for most or almostall other loci. The last backcross generation would be selfed to givepure breeding progeny for the trait being transferred.

[0010] A single cross hybrid corn variety is the cross of two inbredplants, each of which has a genotype which complements the genotype ofthe other. The hybrid progeny of the first generation is designated F₁.Typically, F₁ hybrids are more vigorous than their inbred parents. Thishybrid vigor, or heterosis, is manifested in many polygenic traits,including markedly improved yields, better stalks, better roots, betteruniformity and better insect and disease resistance. In the developmentof hybrids only the F₁ hybrid plants are typically sought. An F₁ singlecross hybrid is produced when two inbred plants are crossed. A doublecross hybrid is produced from four inbred plants crossed in pairs (A×Band C×D) and then the two F₁ hybrids are crossed again (A×B)×(C×D).

[0011] The development of a hybrid corn variety involves three steps:(1) the selection of plants from various germplasm pools; (2) theselfing of the selected plants for several generations to produce aseries of inbred plants, which, although different from each other, eachbreed true and are highly uniform; and (3) crossing the selected inbredplants with unrelated inbred plants to produce the hybrid progeny (F₁).During the inbreeding process in corn, the vigor of the plantsdecreases. Vigor is restored when two unrelated inbred plants arecrossed to produce the hybrid progeny (F₁). An important consequence ofthe homozygosity and homogeneity of the inbred plants is that the hybridbetween any two inbreds is always the same. Once the inbreds that give asuperior hybrid have been identified, hybrid seed can be reproducedindefinitely as long as the homogeneity of the inbred parents ismaintained. Conversely, much of the hybrid vigor exhibited by F₁ hybridsis lost in the next generation (F₂). Consequently, seed from hybridvarieties is not used for planting stock. It is not generally beneficialfor farmers to save seed of F₁ hybrids. Rather, farmers purchase F₁hybrid seed for planting every year.

[0012] North American farmers plant tens of millions of acres of corn atthe present time and there are extensive national and internationalcommercial corn breeding programs. A continuing goal of these cornbreeding programs is to develop corn hybrids that are based on stableinbred plants and have one or more desirable characteristics. Toaccomplish this goal, the corn breeder must select and develop superiorinbred parental plants.

SUMMARY OF THE INVENTION

[0013] In one aspect, the present invention provides a corn plantdesignated 3323. Also provided are corn plants having all thephysiological and morphological characteristics of corn plant 3323. Theinbred corn plant of the invention may further comprise, or have, acytoplasmic or nuclear factor that is capable of conferring malesterility. Parts of the corn plant of the present invention are alsoprovided, for example, pollen obtained from an inbred plant and an ovuleof the inbred plant.

[0014] The invention also concerns seed of the corn plant 3323. A sampleof this seed has been deposited under ATCC Accession No. ______. Theinbred corn seed of the invention may be provided as an essentiallyhomogeneous population of inbred corn seed of the corn plant designated3323. Essentially homogeneous populations of inbred seed are those thatconsist essentially of the particular inbred seed, and are generallyfree from substantial numbers of other seed, so that the inbred seedforms between about 90% and about 100% of the total seed, andpreferably, between about 95% and about 100% of the total seed. Mostpreferably, an essentially homogeneous population of inbred corn seedwill contain between about 98.5%, 99%, 99.5% and about 99.9% of inbredseed, as measured by seed grow outs.

[0015] Therefore, in the practice of the present invention, inbred seedgenerally forms at least about 97% of the total seed. However, even if apopulation of inbred corn seed was found, for some reason, to containabout 50%, or even about 20% or 15% of inbred seed, this would still bedistinguished from the small fraction of inbred seed that may be foundwithin a population of hybrid seed, e.g., within a bag of hybrid seed.In such a bag of hybrid seed offered for sale, the Governmentalregulations require that the hybrid seed be at least about 95% of thetotal seed. In the most preferred practice of the invention, the femaleinbred seed that may be found within a bag of hybrid seed will be about1% of the total seed, or less, and the male inbred seed that may befound within a bag of hybrid seed will be negligible, i.e., will be onthe order of about a maximum of 1 per 100,000, and usually less thanthis value.

[0016] The population of inbred corn seed of the invention can furtherbe particularly defined as being essentially free from hybrid seed. Theinbred seed population may be separately grown to provide an essentiallyhomogeneous population of inbred corn plants designated 3323.

[0017] In another aspect of the invention, single locus converted plantsof 3323 are provided. The single transferred locus may preferably be adominant or recessive allele. Preferably, the single transferred locuswill confer such traits as male sterility, yield stability, waxy starch,yield enhancement, industrial usage, herbicide resistance, insectresistance, resistance to bacterial, fungal, nematode or viral disease,male fertility, and enhanced nutritional quality. The single locus maybe a naturally occurring maize gene or a transgene introduced throughgenetic transformation techniques. When introduced throughtransformation, a single locus may comprise one or more transgenesintegrated at a single chromosomal location.

[0018] In yet another aspect of the invention, an inbred corn plantdesignated 3323 is provided, wherein a cytoplasmically-inherited traithas been introduced into said inbred plant. Suchcytoplasmically-inherited traits are passed to progeny through thefemale parent in a particular cross. An exemplarycytoplasmically-inherited trait is the male sterility trait. Acytoplasmically inherited trait may be a naturally occurring maize traitor a trait introduced through genetic transformation techniques.

[0019] In another aspect of the invention, a tissue culture ofregenerable cells of inbred corn plant 3323 is provided. The tissueculture will preferably be capable of regenerating plants capable ofexpressing all of the physiological and morphological characteristics ofthe foregoing inbred corn plant, and of regenerating plants havingsubstantially the same genotype as the foregoing inbred corn plant.Examples of some of the physiological and morphological characteristicsof the inbred corn plant 3323 include characteristics related to yield,maturity, and kernel quality, each of which are specifically disclosedherein. The regenerable cells in such tissue cultures will preferably bederived from embryos, meristematic cells, immature tassels, microspores,pollen, leaves, anthers, roots, root tips, silk, flowers, kernels, ears,cobs, husks, or stalks, or callus or protoplasts derived from thesetissues. Still further, the present invention provides corn plantsregenerated from the tissue cultures of the invention, the plants havingall the physiological and morphological characteristics of corn plant3323.

[0020] In yet another aspect of the invention, processes are providedfor producing corn seeds or plants, which processes generally comprisecrossing a first parent corn plant with a second parent corn plant,wherein at least one of the first or second parent corn plants is theinbred corn plant designated 3323. These processes may be furtherexemplified as processes for preparing hybrid corn seed or plants,wherein a first inbred corn plant is crossed with a second, distinctinbred corn plant to provide a hybrid that has, as one of its parents,the inbred corn plant 3323 In these processes, the step of crossing willresult in the production of seed. The seed production occurs regardlessof whether the seed is collected or not.

[0021] In a preferred embodiment of the invention, crossing comprisesplanting in pollinating proximity seeds of a first and second parentcorn plant, and preferably, seeds of a first inbred corn plant and asecond, distinct inbred corn plant; cultivating or growing the seeds ofsaid first and second parent corn plants into plants that bear flowers;emasculating the male flowers of the first or second parent corn plant,(i.e., treating or manipulating the flowers so as to prevent pollenproduction, in order to produce an emasculated parent corn plant)allowing natural cross-pollination to occur between the first and secondparent corn plants; and harvesting the seeds from the emasculated parentcorn plant. Where desired, the harvested seed is grown to produce a cornplant or hybrid corn plant.

[0022] The present invention also provides corn seed and plants producedby a process that comprises crossing a first parent corn plant with asecond parent corn plant, wherein at least one of the first or secondparent corn plants is the inbred corn plant designated 3323. In oneembodiment of the invention, corn plants produced by the process arefirst generation (F₁) hybrid corn plants produced by crossing an inbredin accordance with the invention with another, distinct inbred. Thepresent invention further contemplates seed of an F₁ hybrid corn plant.Therefore, certain exemplary embodiments of the invention provide an F₁hybrid corn plant and seed thereof. An example of such a hybrid whichcan be produced with the inbred designated 3323 is the hybrid corn plantdesignated 0993357.

[0023] In still yet another aspect of the invention, an inbred geneticcomplement of the corn plant designated 3323 is provided. The phrase“genetic complement” is used to refer to the aggregate of nucleotidesequences, the expression of which sequences defines the phenotype of,in the present case, a corn plant, or a cell or tissue of that plant. Aninbred genetic complement thus represents the genetic make up of aninbred cell, tissue or plant, and a hybrid genetic complement representsthe genetic make up of a hybrid cell, tissue or plant. The inventionthus provides corn plant cells that have a genetic complement inaccordance with the inbred corn plant cells disclosed herein, andplants, seeds and diploid plants containing such cells.

[0024] Plant genetic complements may be assessed by genetic markerprofiles, and by the expression of phenotypic traits that arecharacteristic of the expression of the genetic complement, e.g.,isozyme typing profiles. Thus, such corn plant cells may be defined ashaving an SSR genetic marker profile in accordance with the profileshown in Table 6, or a genetic isozyme typing profile in accordance withthe profile shown in Table 7, or having both an SSR genetic markerprofile and a genetic isozyme typing profile in accordance with theprofiles shown in Table 6 and Table 7. It is understood that 3323 couldalso be identified by other types of genetic markers such as, forexample, Simple Sequence Length Polymorphisms (SSLPs) (Williams et al.,1990), Randomly Amplified Polymorphic DNAs (RAPDs), DNA AmplificationFingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs),Arbitrary Primed Polymerase Chain Reaction (AP-PCR), Amplified FragmentLength Polymorphisms (AFLPs) (EP 534 858, specifically incorporatedherein by reference in its entirety), and Single NucleotidePolymorphisms (SNPs) (Wang et al., 1998).

[0025] In still yet another aspect, the present invention provideshybrid genetic complements, as represented by corn plant cells, tissues,plants, and seeds, formed by the combination of a haploid geneticcomplement of an inbred corn plant of the invention with a haploidgenetic complement of a second corn plant, preferably, another, distinctinbred corn plant. In another aspect, the present invention provides acorn plant regenerated from a tissue culture that comprises a hybridgenetic complement of this invention.

DETAILED DESCRIPTION OF THE INVENTION

[0026] I. DEFINITIONS OF PLANT CHARACTERISTICS

[0027] Barren Plants: Plants that are barren, i.e., lack an ear withgrain, or have an ear with only a few scattered kernels.

[0028] Cg: Colletotrichum graminicola rating. Rating times 10 isapproximately equal to percent total plant infection.

[0029] CLN: Corn Lethal Necrosis (combination of Maize Chlorotic MottleVirus and Maize Dwarf Mosaic virus) rating: numerical ratings are basedon a severity scale where 1=most resistant to 9=susceptible.

[0030] Cn: Corynebacterium nebraskense rating. Rating times 10 isapproximately equal to percent total plant infection.

[0031] Cz: Cercospora zeae-maydis rating. Rating times 10 isapproximately equal to percent total plant infection.

[0032] Dgg: Diatraea grandiosella girdling rating (values are percentplants girdled and stalk lodged).

[0033] Dropped Ears: Ears that have fallen from the plant to the ground.

[0034] Dsp: Diabrotica species root ratings (1=least affected to9=severe pruning).

[0035] Ear-Attitude: The attitude or position of the ear at harvestscored as upright, horizontal, or pendant.

[0036] Ear-Cob Color: The color of the cob, scored as white, pink, red,or brown.

[0037] Ear-Cob Diameter: The average diameter of the cob measured at themidpoint.

[0038] Ear-Cob Strength: A measure of mechanical strength of the cobs tobreakage, scored as strong or weak.

[0039] Ear-Diameter: The average diameter of the ear at its midpoint.

[0040] Ear-Dry Husk Color: The color of the husks at harvest scored asbuff, red, or purple.

[0041] Ear-Fresh Husk Color: The color of the husks 1 to 2 weeks afterpollination scored as green, red, or purple.

[0042] Ear-Husk Bract: The length of an average husk leaf scored asshort, medium, or long.

[0043] Ear-Husk Cover: The average distance from the tip of the ear tothe tip of the husks. Minimum value no less than zero.

[0044] Ear-Husk Opening: An evaluation of husk tightness at harvestscored as tight, intermediate, or open.

[0045] Ear-Length: The average length of the ear.

[0046] Ear-Number Per Stalk: The average number of ears per plant.

[0047] Ear-Shank Internodes: The average number of internodes on the earshank.

[0048] Ear-Shank Length: The average length of the ear shank.

[0049] Ear-Shelling Percent: The average of the shelled grain weightdivided by the sum of the shelled grain weight and cob weight for asingle ear.

[0050] Ear-Silk Color: The color of the silk observed 2 to 3 days aftersilk emergence scored as green-yellow, yellow, pink, red, or purple.

[0051] Ear-Taper (Shape): The taper or shape of the ear scored asconical, semi-conical, or cylindrical.

[0052] Ear-Weight: The average weight of an ear.

[0053] Early Stand: The percent of plants that emerge from the ground asdetermined in the early spring.

[0054] ER: Ear rot rating (values approximate percent ear rotted).

[0055] Final Stand Count: The number of plants just prior to harvest.

[0056] GDUs: Growing degree units which are calculated by the BargerMethod, where the heat units for a 24-h period are calculated as GDUs=[(Maximum daily temperature +Minimum daily temperature)/2] -50. Thehighest maximum daily temperature used is 86° F. and the lowest minimumtemperature used is 50° F.

[0057] GDUs to Shed: The number of growing degree units (GDUs) or heatunits required for an inbred line or hybrid to have approximately 50% ofthe plants shedding pollen as measured from time of planting. GDUs toshed is determined by summing the individual GDU daily values fromplanting date to the date of 50% pollen shed.

[0058] GDUs to Silk: The number of growing degree units for an inbredline or hybrid to have approximately 50% of the plants with silkemergence as measured from time of planting. GDUs to silk is determinedby summing the individual GDU daily values from planting date to thedate of 50% silking.

[0059] Hc2: Helminthosporium carbonum race 2 rating. Rating times 10 isapproximately equal to percent total plant infection.

[0060] Hc3: Helminthosporium carbonum race 3 rating. Rating times 10 isapproximately equal to percent total plant infection.

[0061] Hm: Helminthosporium maydis race 0 rating. Rating times 10 isapproximately equal to percent total plant infection.

[0062] Ht1: Helminthosporium turcicum race 1 rating. Rating times 10 isapproximately equal to percent total plant infection.

[0063] Ht2: Helminthosporium turcicum race 2 rating. Rating times 10 isapproximately equal to percent total plant infection.

[0064] HtG: Chlorotic-lesion type resistance. += indicates the presenceof Ht chlorotic-lesion type resistance; −=indicates absence of Htchlorotic-lesion type resistance; and +/−= indicates segregation of Htchlorotic-lesion type resistance. Rating times 10 is approximately equalto percent total plant infection.

[0065] Kernel-Aleurone Color: The color of the aleurone scored as white,pink, tan, brown, bronze, red, purple, pale purple, colorless, orvariegated.

[0066] Kernel-Cap Color: The color of the kernel cap observed at drystage, scored as white, lemon-yellow, yellow, or orange.

[0067] Kernel-Endosperm Color: The color of the endosperm scored aswhite, pale yellow, or yellow.

[0068] Kernel-Endosperm Type: The type of endosperm scored as normal,waxy, or opaque.

[0069] Kernel-Grade: The percent of kernels that are classified asrounds.

[0070] Kernel-Length: The average distance from the cap of the kernel tothe pedicel.

[0071] Kernel-Number Per Row: The average number of kernels in a singlerow.

[0072] Kernel-Pericarp Color: The color of the pericarp scored ascolorless, red-white crown, tan, bronze, brown, light red, cherry red,or variegated.

[0073] Kernel-Row Direction: The direction of the kernel rows on the earscored as straight, slightly curved, spiral, or indistinct (scattered).

[0074] Kernel-Row Number: The average number of rows of kernels on asingle ear.

[0075] Kernel-Side Color: The color of the kernel side observed at thedry stage, scored as white, pale yellow, yellow, orange, red, or brown.

[0076] Kernel-Thickness: The distance across the narrow side of thekernel.

[0077] Kernel-Type: The type of kernel scored as dent, flint, orintermediate.

[0078] Kernel-Weight: The average weight of a predetermined number ofkernels.

[0079] Kernel-Width: The distance across the flat side of the kernel.

[0080] Kz: Kabatiella zeae rating. Rating times 10 is approximatelyequal to percent total plant infection.

[0081] Leaf-Angle: Angle of the upper leaves to the stalk scored asupright (0 to 30 degrees), intermediate (30 to 60 degrees), or lax (60to 90 degrees).

[0082] Leaf-Color: The color of the leaves 1 to 2 weeks afterpollination scored as light green, medium green, dark green, or verydark green.

[0083] Leaf-Length: The average length of the primary ear leaf.

[0084] Leaf-Longitudinal Creases: A rating of the number of longitudinalcreases on the leaf surface 1 to 2 weeks after pollination. Creases arescored as absent, few, or many.

[0085] Leaf-Marginal Waves: A rating of the waviness of the leaf margin1 to 2 weeks after pollination. Rated as none, few, or many.

[0086] Leaf-Number: The average number of leaves of a mature plant.Counting begins with the cotyledonary leaf and ends with the flag leaf

[0087] Leaf-Sheath Anthocyanin: A rating of the level of anthocyanin inthe leaf sheath 1 to 2 weeks after pollination, scored as absent,basal-weak, basal-strong, weak or strong.

[0088] Leaf-Sheath Pubescence: A rating of the pubescence of the leafsheath. Ratings are taken 1 to 2 weeks after pollination and scored aslight, medium, or heavy.

[0089] Leaf-Width: The average width of the primary ear leaf measured atits widest point.

[0090] LSS: Late season standability (values times 10 approximatepercent plants lodged in disease evaluation plots).

[0091] Moisture: The moisture of the grain at harvest.

[0092] On1: Ostrinia nubilalis 1st brood rating (1=resistant to9=susceptible).

[0093] On2: Ostrinia nubilalis 2nd brood rating (1=resistant to9=susceptible).

[0094] Relative Maturity: A maturity rating based on regressionanalysis. The regression analysis is developed by utilizing checkhybrids and their previously established day rating versus actualharvest moistures. Harvest moisture on the hybrid in question isdetermined and that moisture value is inserted into the regressionequation to yield a relative maturity.

[0095] Root Lodging: Root lodging is the percentage of plants that rootlodge. A plant is counted as root lodged if a portion of the plant leansfrom the vertical axis by approximately 30 degrees or more.

[0096] Seedling Color: Color of leaves at the 6 to 8 leaf stage.

[0097] Seedling Height: Plant height at the 6 to 8 leaf stage.

[0098] Seedling Vigor: A visual rating of the amount of vegetativegrowth on a 1 to 9 scale, where 1 equals best. The score is taken whenthe average entry in a trial is at the fifth leaf stage.

[0099] Selection Index: The selection index gives a single measure ofhybrid's worth based on information from multiple traits. One of thetraits that is almost always included is yield. Traits may be weightedaccording to the level of importance assigned to them.

[0100] Sr: Sphacelotheca reiliana rating is actual percent infection.

[0101] Stalk-Anthocyanin: A rating of the amount of anthocyaninpigmentation in the stalk. The stalk is rated 1 to 2 weeks afterpollination as absent, basal-weak, basal-strong, weak, or strong.

[0102] Stalk-Brace Root Color: The color of the brace roots observed 1to 2 weeks after pollination as green, red, or purple.

[0103] Stalk-Diameter: The average diameter of the lowest visibleinternode of the stalk.

[0104] Stalk-Ear Height: The average height of the ear measured from theground to the point of attachment of the ear shank of the top developedear to the stalk.

[0105] Stalk-Internode Direction: The direction of the stalk internodeobserved after pollination as straight or zigzag.

[0106] Stalk-Internode Length: The average length of the internode abovethe primary ear.

[0107] Stalk Lodging: The percentage of plants that did stalk lodge.Plants are counted as stalk lodged if the plant is broken over or offbelow the ear.

[0108] Stalk-Nodes With Brace Roots: The average number of nodes havingbrace roots per plant.

[0109] Stalk-Plant Height: The average height of the plant as measuredfrom the soil to the tip of the tassel.

[0110] Stalk-Tillers: The percent of plants that have tillers. A tilleris defined as a secondary shoot that has developed as a tassel capableof shedding pollen.

[0111] Staygreen: Staygreen is a measure of general plant health nearthe time of black layer formation (physiological maturity). It isusually recorded at the time the ear husks of most entries within atrial have turned a mature color. Scoring is on a 1 to 9 basis where 1equals best.

[0112] STR: Stalk rot rating (values represent severity rating of 1=25%of inoculated internode rotted to 9=entire stalk rotted and collapsed).

[0113] SVC: Southeastern Virus Complex (combination of Maize ChloroticDwarf Virus and Maize Dwarf Mosaic Virus) rating; numerical ratings arebased on a severity scale where 1=most resistant to 9=susceptible (1988reactions are largely Maize Dwarf Mosaic Virus reactions).

[0114] Tassel-Anther Color: The color of the anthers at 50% pollen shedscored as green-yellow, yellow, pink, red, or purple.

[0115] Tassel-Attitude: The attitude of the tassel after pollinationscored as open or compact.

[0116] Tassel-Branch Angle: The angle of an average tassel branch to themain stem of the tassel scored as upright (less than 30 degrees),intermediate (30 to 45 degrees), or lax (greater than 45 degrees).

[0117] Tassel-Branch Number: The average number of primary tasselbranches.

[0118] Tassel-Glume Band: The closed anthocyanin band at the base of theglume scored as present or absent.

[0119] Tassel-Glume Color: The color of the glumes at 50% shed scored asgreen, red, or purple.

[0120] Tassel-Length: The length of the tassel measured from the base ofthe bottom tassel branch to the tassel tip.

[0121] Tassel-Peduncle Length: The average length of the tasselpeduncle, measured from the base of the flag leaf to the base of thebottom tassel branch.

[0122] Tassel-Pollen Shed: A visual rating of pollen shed determined bytapping the tassel and observing the pollen flow of approximately fiveplants per entry. Rated on a 1 to 9 scale where 9 =sterile, 1=mostpollen.

[0123] Tassel-Spike Length: The length of the spike measured from thebase of the top tassel branch to the tassel tip.

[0124] Test Weight: Weight of the grain in pounds for a given volume(bushel) adjusted to 15.5% moisture.

[0125] Yield: Yield of grain at harvest adjusted to 15.5% moisture.

[0126] II. OTHER DEFINITIONS

[0127] Allele: Any of one or more alternative forms of a gene locus, allof which alleles relate to one trait or characteristic. In a diploidcell or organism, the two alleles of a given gene occupy correspondingloci on a pair of homologous chromosomes.

[0128] Backcrossing: A process in which a breeder repeatedly crosseshybrid progeny back to one of the parents, for example, a firstgeneration hybrid (F₁) with one of the parental genotypes of the F₁hybrid.

[0129] Chromatography: A technique wherein a mixture of dissolvedsubstances are bound to a solid support followed by passing a column offluid across the solid support and varying the composition of the fluid.The components of the mixture are separated by selective elution.

[0130] Crossing: The pollination of a female flower of a corn plant,thereby resulting in the production of seed from the flower.

[0131] Cross-pollination: Fertilization by the union of two gametes fromdifferent plants.

[0132] Diploid: A cell or organism having two sets of chromosomes.

[0133] Electrophoresis: A process by which particles suspended in afluid or a gel matrix are moved under the action of an electrical field,and thereby separated according to their charge and molecular weight.This method of separation is well known to those skilled in the art andis typically applied to separating various forms of enzymes and of DNAfragments produced by restriction endonucleases.

[0134] Emasculate: The removal of plant male sex organs or theinactivation of the organs with a chemical agent or a cytoplasmic ornuclear genetic factor conferring male sterility.

[0135] Enzymes: Molecules which can act as catalysts in biologicalreactions.

[0136] F₁ Hybrid: The first generation progeny of the cross of twoplants.

[0137] Genetic Complement: An aggregate of nucleotide sequences, theexpression of which sequences defines the phenotype in corn plants, orcomponents of plants including cells or tissue.

[0138] Genotype: The genetic constitution of a cell or organism.

[0139] Haploid: A cell or organism having one set of the two sets ofchromosomes in a diploid.

[0140] Isozymes: Detectable variants of an enzyme, the variantscatalyzing the same reaction(s) but differing from each other, e.g., inprimary structure and/or electrophoretic mobility. The differencesbetween isozymes are under single gene, codominant control.Consequently, electrophoretic separation to produce band patterns can beequated to different alleles at the DNA level. Structural differencesthat do not alter charge cannot be detected by this method.

[0141] Isozyme typing profile: A profile of band patterns of isozymesseparated by electrophoresis that can be equated to different alleles atthe DNA level.

[0142] Linkage: A phenomenon wherein alleles on the same chromosome tendto segregate together more often than expected by chance if theirtransmission was independent.

[0143] Marker: A readily detectable phenotype, preferably inherited incodominant fashion (both alleles at a locus in a diploid heterozygoteare readily detectable), with no environmental variance component, i.e.,heritability of 1.

[0144] 3323: The corn plant from which seeds having ATCC Accession No.______ were obtained, as well as plants grown from those seeds.

[0145] Phenotype: The detectable characteristics of a cell or organism,which characteristics are the manifestation of gene expression.

[0146] Quantitative Trait Loci (QTL): Genetic loci that contribute, atleast in part, certain numerically representable traits that are usuallycontinuously distributed.

[0147] Regeneration: The development of a plant from tissue culture.

[0148] SSR genetic marker profile: A profile of simple sequence repeatsscored by gel electrophoresis following PCR™ amplification usingflanking oligonucleotide primers.

[0149] Self-pollination: The transfer of pollen from the anther to thestigma of the same plant.

[0150] Single Locus Converted (Conversion) Plant: Plants which aredeveloped by a plant breeding technique called backcrossing whereinessentially all of the desired morphological and physiologicalcharacteristics of an inbred are recovered in addition to thecharacteristics conferred by the single locus transferred into theinbred via the backcrossing technique. A single locus may comprise onegene, or in the case of transgenic plants, one or more transgenesintegrated into the host genome at a single site (locus).

[0151] Tissue Culture: A composition comprising isolated cells of thesame or a different type or a collection of such cells organized intoparts of a plant.

[0152] Transgene: A genetic sequence which has been introduced into thenuclear or chloroplast genome of a maize plant by a genetictransformation technique.

[0153] The following examples are included to demonstrate preferredembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples that followrepresent techniques discovered by the inventor to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments that are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

[0154] III. INBRED CORN PLANT 3323

[0155] In accordance with one aspect of the present invention, there isprovided a novel inbred corn plant, designated 3323. In accordance withone aspect of the present invention, there is provided a novel inbredcorn plant, designated 3323. Inbred corn plant 3323 can be compared toinbred corn plants 3327 and 5750. 3327 differs significantly (at the 1%,5%, or 10% level) from these inbred lines in several aspects (Table 1and Table 2). TABLE 1 Comparison of 3323 with 3327 3323 3327 DIFF PVALUE BARREN % 0.9 4.5 −3.6 0.006* EHT INCH 27.3 25.3 2.0 0.884 FINAL63.2 63.4 −0.2 0.248 MST % 21.3 19.8 1.5 0.972 PHT INCH 77.3 78.1 −0.80.562 SHED GDU 1424.4 1409.0 15.4 0.838 SILK GDU 1428.4 1467.2 −38.80.619 STL % 0.7 2.5 −1.8 0.000** YLD BU/A 96.6 60.6 36.0 0.286Significance Levels are indicated as: + = 10%, * = 5%, ** = 1%. LegendAbbreviations: BARREN % = Barren Plants (percent) EHT INCH = Ear Height(inches) FINAL = Final Stand MST % = Moisture (percent) PHT INCH = PlantHeight (inches) SHED GDU = GDUs to Shed SILK GDU = GDUs to Silk STL % =Stalk Lodging (percent) YLD BU/A = Yield (bushels/acre)

[0156] TABLE 2 Comparison of 3323 with 5750 3323 5750 DIFF P VALUEBARREN % 0.9 2.7 −1.8 0.045* EHT INCH 27.3 25.4 1.9 0.182 FINAL 63.263.9 −0.7 0.661 MST % 21.3 20.8 0.5 0.832 PHT INCH 77.3 73.3 3.6 0.174SHED GDU 1424.4 1453.7 −29.3 0.804 SILK GDU 1428.4 1470.7 −42.3 0.536STL % 0.7 1.8 −1.1 0.000** YLD BU/A 96.6 70.5 26.1 0.536 SignificanceLevels are indicated as: + = 10%, * = 5% ** = 1%. Legend Abbreviations:BARREN % = Barren Plants (percent) EHT INCH = Ear Height (inches) FINAL= Final Stand MST % = Moisture (percent) PHT INCH = Plant Height(inches) SHED GDU = GDUs to Shed SILK GDU = GDUs to Silk STL % = StalkLodging (percent) YLD BU/A = Yield (bushels/acre)

[0157] A. Origin and Breeding History

[0158] Inbred plant 3323 was derived from the cross between the lines3137 and 5727. The origin and breeding history of inbred plant 3323 canbe summarized as follows: Summer 1993 The inbred line 3137 (aproprietary ASGROW Seed Company inbred) was crossed to the inbred line5727 (a proprietary ASGROW Seed Company inbred) nursery rowsNR93SO:14774 and NR93SO:14765. Winter The S0 seed was grown andself-pollinated (nursery row 1993-94 93KNSELF03:5038) Summer 1994 S1seed was grown and self-pollinated (nursery rows NR94S1IT:26066-27054)Summer 1995 S2 seed was grown and self-pollinated. One ear was selectedfrom ear row selection number 23 in nursery NR9554DL:43718. Winter S3seed was grown ear-to-row and self-pollinated 1995-96 (nursery row95KNIPH:29074. Three ears were selected. Summer 1996 S4 seed was grownear-to-row and self-pollinated (nursery NR96S6UP:51062-67. Winter S5seed was grown and self-pollinated (nursery 1996-97 96KNS200:43223. 5ears were selected. Summer 1997 S6 seed was grown and self-pollinated(nursery NR97S6UP:261114-27106) Winter S7 seed was grown andself-pollinated (nursery 1997-98 97KNS4000IPKN:37256-37263 Summer 1998S8 seed was grown ear-to-row. Final selection was made from nursery rowsNR98S6UP:5123-6119. S9 seed was bulked.

[0159] 3323 shows uniformity and stability within the limits ofenvironmental influence for the traits described hereinafter in Table 3.3323 has been self-pollinated and ear-rowed a sufficient number ofgenerations with careful attention paid to uniformity of plant type toensure homozygosity and phenotypic stability. No variant traits havebeen observed or are expected in 3323.

[0160] Inbred corn plants can be reproduced by planting the seeds of theinbred corn plant 3323, growing the resulting corn plants underself-pollinating or sib-pollinating conditions with adequate isolationusing standard techniques well known to an artisan skilled in theagricultural arts Seeds can be harvested from such a plant usingstandard, well known procedures.

[0161] B. Phenotypic Description

[0162] In accordance with another aspect of the present invention, thereis provided a corn plant having the physiological and morphologicalcharacteristics of corn plant 3323. A description of the physiologicaland morphological characteristics of corn plant 3323, as well ascomparative inbreds 3327 and 5750 is presented in Table 3. TABLE 3Physiological and Morphological Characteristics Traits for the 3323Phenotype and a Comparative Inbred VALUE CHARACTERISTIC 3323 33275750 1. STALK Diameter  2.1  1.9  1.8 (width) cm. Anthocyanin AbsentAbsent Basal-Weak Brace Root Color Absent Absent Absent Nodes With  1.1 1.7  1.6 Brace Roots Internode Straight Straight Straight DirectionInternode  14.9  15.7  15.3 Length cm. 2. LEAF Color Green Green LightGreen Length cm.  69.2  75.7  57.9 Width cm.  8.8  7.6  7.7 SheathAbsent Absent Absent Anthocyanin Sheath Moderate Moderate ModeratePubescence Marginal Waves Moderate Moderate Moderate Longitudinal FewFew Few Creases 3. TASSEL Length cm.  54.3  47.7  37.7 Spike Length cm. 15.9  19.7  18.9 Peduncle  9.0  12.8  6.7 Length cm. Branch Number  6.6 4.7  4.5 Anther Color Green- Green-Yellow Green-Yellow Yellow GlumeColor Green Green Green Glume Band Absent Absent Absent 4. EAR SilkColor Green- Green-Yellow Green-Yellow Yellow Number Per Stalk  1.0  1.0 1.0 Position (attitude) Upright Horizontal Upright Length cm.  14.6 12.5  11.8 Shape Semi-Conical Semi-Conical Semi-Conical Diameter cm. 4.2  4.5  4.0 Shank Length cm.  7.2  8.9  11.2 Husk Bract Short ShortShort Husk Cover cm.  5.3  4.9  10.4 Husk Opening Loose Loose Loose HuskColor Fresh Green Green Green Husk Color Buff Buff Buff Dry Cob Diameter 2.4  2.5  2.1 cm. Cob Color Red Red Red Shelling Percent  86.6  85.8 83.1 5. KERNEL Row Number  13.6  16.4  12.8 Number Per Row  29.4  23.2 22.4 Row Direction Straight Straight Straight Type Dent Semi-DentSemi-Dent Cap Color Orange Orange Yellow Side Color Orange Orange YellowLength  10.8  11.0  12.0 (depth) mm. Width mm.  8.4  8.2  8.6 Thickness 3.6  4.0  3.2 Weight of 331.0 312.0 306.0 1000 K. gm. Endosperm TypeNormal Normal Normal Endosperm Color Yellow Yellow Yellow

[0163] C. DEPOSIT INFORMATION

[0164] A deposit of 2500 seeds of the inbred corn plant designated 3323has been made with the American Type Culture Collection (ATCC), 10801University Blvd., Manassas, Va. on (______ ______,______). Thosedeposited seeds have been assigned ATCC Accession No. ______.The depositwas made in accordance with the terms and provisions of the BudapestTreaty relating to deposit of microorganisms and was made for a term ofat least thirty (30) years and at least five (05) years after the mostrecent request for the furnishing of a sample of the deposit is receivedby the depository, or for the effective term of the patent, whichever islonger, and will be replaced if it becomes non-viable during thatperiod.

[0165] IV. SINGLE LOCUS CONVERSIONS

[0166] When the term inbred corn plant is used in the context of thepresent invention, this also includes any single locus conversions ofthat inbred. The term single locus converted plant as used herein refersto those corn plants which are developed by a plant breeding techniquecalled backcrossing wherein essentially all of the desired morphologicaland physiological characteristics of an inbred are recovered in additionto the single locus transferred into the inbred via the backcrossingtechnique. Backcrossing methods can be used with the present inventionto improve or introduce a characteristic into the inbred. The termbackcrossing as used herein refers to the repeated crossing of a hybridprogeny back to one of the parental corn plants for that inbred. Theparental corn plant which contributes the locus or loci for the desiredcharacteristic is termed the nonrecurrent or donor parent. Thisterminology refers to the fact that the nonrecurrent parent is used onetime in the backcross protocol and therefore does not recur. Theparental corn plant to which the locus or loci from the nonrecurrentparent are transferred is known as the recurrent parent as it is usedfor several rounds in the backcrossing protocol (Poehlman et al., 1995;Fehr, 1987; Sprague and Dudley, 1988). In a typical backcross protocol,the original inbred of interest (recurrent parent) is crossed to asecond inbred (nonrecurrent parent) that carries the single locus ofinterest to be transferred. The resulting progeny from this cross arethen crossed again to the recurrent parent and the process is repeateduntil a corn plant is obtained wherein essentially all of the desiredmorphological and physiological characteristics of the recurrent parentare recovered in the converted plant, in addition to the singletransferred locus from the nonrecurrent parent. The backcross processmay be accelerated by the use of genetic markers, such as SSR, RFLP, SNPor AFLP markers to identify plants with the greatest genetic complementfrom the recurrent parent.

[0167] The selection of a suitable recurrent parent is an important stepfor a successful backcrossing procedure. The goal of a backcrossprotocol is to alter or substitute a single trait or characteristic inthe original inbred. To accomplish this, a single locus of the recurrentinbred is modified or substituted with the desired locus from thenonrecurrent parent, while retaining essentially all of the rest of thedesired genetic, and therefore the desired physiological andmorphological constitution of the original inbred. The choice of theparticular nonrecurrent parent will depend on the purpose of thebackcross; one of the major purposes is to add some commerciallydesirable, agronomically important trait to the plant. The exactbackcrossing protocol will depend on the characteristic or trait beingaltered to determine an appropriate testing protocol. Althoughbackcrossing methods are simplified when the characteristic beingtransferred is a dominant allele, a recessive allele may also betransferred. In this instance it may be necessary to introduce a test ofthe progeny to determine if the desired characteristic has beensuccessfully transferred.

[0168] Many single locus traits have been identified that are notregularly selected for in the development of a new inbred but that canbe improved by backcrossing techniques. Single locus traits may or maynot be transgenic; examples of these traits include, but are not limitedto, male sterility, waxy starch, herbicide resistance, resistance forbacterial, fungal, or viral disease, insect resistance, male fertility,enhanced nutritional quality, industrial usage, yield stability, andyield enhancement. These genes are generally inherited through thenucleus, but may be inherited through the cytoplasm. Some knownexceptions to this are genes for male sterility, some of which areinherited cytoplasmically, but still act as single locus traits. Anumber of exemplary single locus traits are described in, for example,PCT Application WO 95/06128, the disclosure of which is specificallyincorporated herein by reference.

[0169] Examples of genes conferring male sterility include thosedisclosed in U.S. Pat. No. 3,861,709, U.S. Pat. No. 3,710,511, U.S. Pat.No. 4,654,465, U.S. Pat. No. 5,625,132, and U.S. Pat. No. 4,727,219,each of the disclosures of which are specifically incorporated herein byreference in their entirety. A particularly useful type of malesterility gene is one which can be induced by exposure to a chemicalagent, for example, a herbicide (U.S. Patent Ser. No. 08/927,368, filedSep. 11, 1997, the disclosure of which is specifically incorporatedherein by reference in its entirety). Both inducible and non-induciblemale sterility genes can increase the efficiency with which hybrids aremade, in that they eliminate the need to physically emasculate the cornplant used as a female in a given cross.

[0170] Where one desires to employ male-sterility systems with a cornplant in accordance with the invention, it may be beneficial to alsoutilize one or more male-fertility restorer genes. For example, wherecytoplasmic male sterility (CMS) is used, hybrid seed productionrequires three inbred lines: (1) a cytoplasmically male-sterile linehaving a CMS cytoplasm; (2) a fertile inbred with normal cytoplasm,which is isogenic with the CMS line for nuclear genes (“maintainerline”); and (3) a distinct, fertile inbred with normal cytoplasm,carrying a fertility restoring gene (“restorer” line). The CMS line ispropagated by pollination with the maintainer line, with all of theprogeny being male sterile, as the CMS cytoplasm is derived from thefemale parent. These male sterile plants can then be efficientlyemployed as the female parent in hybrid crosses with the restorer line,without the need for physical emasculation of the male reproductiveparts of the female parent.

[0171] The presence of a male-fertility restorer gene results in theproduction of fully fertile F₁ hybrid progeny. If no restorer gene ispresent in the male parent, male-sterile hybrids are obtained. Suchhybrids are useful where the vegetative tissue of the corn plant isutilized, e.g., for silage, but in most cases, the seeds will be deemedthe most valuable portion of the crop, so fertility of the hybrids inthese crops must be restored. Therefore, one aspect of the currentinvention concerns the inbred corn plant 3323 comprising a single genecapable of restoring male fertility in an otherwise male-sterile inbredor hybrid plant. Examples of male-sterility genes and correspondingrestorers which could be employed with the inbred of the invention arewell known to those of skill in the art of plant breeding and aredisclosed in, for instance, U.S. Pat. No. 5,530,191; U.S. Pat. No.5,689,041; U.S. Pat. No. 5,741,684; and U.S. Pat. No. 5,684,242, thedisclosures of which are each specifically incorporated herein byreference in their entirety.

[0172] Direct selection may be applied where a single locus acts as adominant trait. An example of a dominant trait is the herbicideresistance trait. For this selection process, the progeny of the initialcross are sprayed with the herbicide prior to the backcrossing. Thespraying eliminates any plants which do not have the desired herbicideresistance characteristic, and only those plants which have theherbicide resistance gene are used in the subsequent backcross. Thisprocess is then repeated for all additional backcross generations.

[0173] Many useful single locus traits are those which are introduced bygenetic transformation techniques. Methods for the genetictransformation of maize are known to those of skill in the art. Forexample, methods which have been described for the genetictransformation of maize include electroporation (U.S. Pat. No.5,384,253), electrotransformation (U.S. Pat. No. 5,371,003),microprojectile bombardment (U.S. Pat. No. 5,550,318; U.S. Pat. No.5,736,369, U.S. Pat. No. 5,538,880; and PCT Publication WO 95/06128),Agrobacterium-mediated transformation (U.S. Pat. No. 5,591,616 and E.P.Publication EP672752), direct DNA uptake transformation of protoplasts(Omirulleh et al., 1993) and silicon carbide fiber-mediatedtransformation (U.S. Pat. No. 5,302,532 and U.S. Pat. No. 5,464,765).

[0174] A type of single locus trait which can be introduced by genetictransformation (U S. Pat. No. 5,554,798) and has particular utility is agene which confers resistance to the herbicide glyphosate. Glyphosateinhibits the action of the enzyme EPSPS, which is active in thebiosynthetic pathway of aromatic amino acids. Inhibition of this enzymeleads to starvation for the amino acids phenylalanine, tyrosine, andtryptophan and secondary metabolites derived therefrom. Mutants of thisenzyme are available which are resistant to glyphosate. For example,U.S. Pat. No. 4,535,060 describes the isolation of EPSPS mutations whichconfer glyphosate resistance upon organisms having the Salmonellatyphimurium gene for EPSPS, aroA. A mutant EPSPS gene having similarmutations has also been cloned from Zea mays. The mutant gene encodes aprotein with amino acid changes at residues 102 and 106 (PCT PublicationWO 97/04103). When a plant comprises such a gene, a herbicide resistantphenotype results.

[0175] Plants having inherited a transgene comprising a mutated EPSPSgene may, therefore, be directly treated with the herbicide glyphosatewithout the result of significant damage to the plant. This phenotypeprovides farmers with the benefit of controlling weed growth in a fieldof plants having the herbicide resistance trait by application of thebroad spectrum herbicide glyphosate. For example, one could apply theherbicide ROUNDUP™, a commercial formulation of glyphosate manufacturedand sold by the Monsanto Company, over the top in fields whereglyphosate resistant corn plants are grown. The herbicide applicationrates may typically range from 4 ounces of ROUNDUP™ to 256 ouncesROUNDUP™ per acre. More preferably, about 16 ounces to about 64 ouncesper acre of ROUNDUP™ may be applied to the field. However, theapplication rate may be increased or decreased as needed, based on theabundance and I or type of weeds being treated. Additionally, dependingon the location of the field and weather conditions, which willinfluence weed growth and the type of weed infestation, it may bedesirable to conduct further glyphosate treatments. The secondglyphosate application will also typically comprise an application rateof about 16 ounces to about 64 ounces of ROUNDUP™ per acre treated.Again, the treatment rate may be adjusted based on field conditions.Such methods of application of herbicides to agricultural crops are wellknown in the art and are summarized in general in Anderson (1983).

[0176] Alternatively, more than one single locus trait may beintrogressed into an elite inbred by the method of backcross conversion.A selectable marker gene and a gene encoding a protein which confers atrait of interest may be simultaneously introduced into a maize plant asa result of genetic transformation. Usually one or more introduced geneswill integrate into a single chromosome site in the host cell's genome.For example, a selectable marker gene encoding phosphinothricin acetyltransferase (PPT) (e.g., a bar gene) and conferring resistance to theactive ingredient in some herbicides by inhibiting glutamine synthetase,and a gene encoding an endotoxin from Bacillus thuringiensis (Bt) andconferring resistance to particular classes of insects, e.g.,lepidopteran insects, in particular the European Corn Borer, may besimultaneously introduced into a host genome. Furthermore, through theprocess of backcross conversion more than one transgenic trait may betransferred into an elite inbred.

[0177] The waxy characteristic is an example of a recessive trait. Inthis example, the progeny resulting from the first backcross generation(BC1) must be grown and selfed. A test is then run on the selfed seedfrom the BC1 plant to determine which BC1 plants carried the recessivegene for the waxy trait. In other recessive traits additional progenytesting, for example growing additional generations such as the BC1S1,may be required to determine which plants carry the recessive gene.

[0178] V. ORIGIN AND BREEDING HISTORY OF AN EXEMPLARY SINGLE LOCUSCONVERTED PLANT

[0179] 85DGD1 MLms is a single locus conversion of 85DGD1 to cytoplasmicmale sterility. 85DGD1 MLms was derived using backcross methods. 85DGD1(a proprietary inbred of DEKALB Genetics Corporation) was used as therecurrent parent and MLms, a germplasm source carrying ML cytoplasmicsterility, was used as the nonrecurrent parent. The breeding history ofthe single locus converted inbred 85DGD1 MLms can be summarized asfollows: Hawaii Nurseries Made up S-O: Female row 585 male row PlantingDate 04-02-1992 500 Hawaii Nurseries S-O was grown and plants werePlanting Date 07-15-1992 backcrossed times 85DGD1 (rows 444 ′ 443)Hawaii Nurseries Bulked seed of the BC1 was grown and Planting Date11-18-1992 backcrossed times 85DGD1 (rows V3-27 ′ V3-26) HawaiiNurseries Bulked seed of the BC2 was grown and Planting Date 04-02-1993backcrossed times 85DGD1 (rows 37 ′ 36) Hawaii Nurseries Bulked seed ofthe BC3 was grown and Planting Date 07-14-1993 backcrossed times 85DGD1(rows 99 ′ 98) Hawaii Nurseries Bulked seed of BC4 was grown andPlanting Date 10-28-1993 backcrossed times 85DGD1 (rows KS-63 ′ KS-62)Summer 1994 A single ear of the BC5 was grown and backcrossed times85DGD1 (MC94-822 ′ MC94-822-7) Winter 1994 Bulked seed of the BC6 wasgrown and backcrossed times 85DGD1 (3Q-1 ′ 3Q-2) Summer 1995 Seed of theBC7 was bulked and named 85DGD1 MLms.

[0180] VI. TISSUE CULTURES AND IN VITRO REGENERATION OF CORN PLANTS

[0181] A further aspect of the invention relates to tissue cultures ofthe corn plant designated 3323. As used herein, the term “tissueculture” indicates a composition comprising isolated cells of the sameor a different type or a collection of such cells organized into partsof a plant. Exemplary types of tissue cultures are protoplasts, calliand plant cells that are intact in plants or parts of plants, such asembryos, pollen, flowers, kernels, ears, cobs, leaves, husks, stalks,roots, root tips, anthers, silk, and the like. In a preferredembodiment, the tissue culture comprises embryos, protoplasts,meristematic cells, pollen, leaves or anthers derived from immaturetissues of these plant parts. Means for preparing and maintaining planttissue cultures are well known in the art (U.S. Pat. No. 5,538,880; andU.S. Pat. No. 5,550,318, each incorporated herein by reference in theirentirety). By way of example, a tissue culture comprising organs such astassels or anthers has been used to produce regenerated plants (U.S.Pat. No. 5,445,961 and U.S. Pat. No. 5,322,789; the disclosures of whichare incorporated herein by reference).

[0182] VII. TASSEL/ANTHER CULTURE

[0183] Tassels contain anthers which in turn enclose microspores.Microspores develop into pollen. For anther/microspore culture, iftassels are the plant composition, they are preferably selected at astage when the microspores are uninucleate, that is, include only one,rather than 2 or 3 nuclei. Methods to determine the correct stage arewell known to those skilled in the art and include mitramycinfluorescent staining (Pace et aL, 1987), trypan blue (preferred) andacetocarmine squashing. The mid-uninucleate microspore stage has beenfound to be the developmental stage most responsive to the subsequentmethods disclosed to ultimately produce plants.

[0184] Although microspore-containing plant organs such as tassels cangenerally be pretreated at any cold temperature below about 25° C., arange of 4 to 25° C. is preferred, and a range of 8 to 14° C. isparticularly preferred. Although other temperatures yield embryoids andregenerated plants, cold temperatures produce optimum response ratescompared to pretreatment at temperatures outside the preferred range.Response rate is measured as either the number of embryoids or thenumber of regenerated plants per number of microspores initiated inculture. Exemplary methods of microspore culture are disclosed in, forexample, U.S. Pat. No. 5,322,789 and U.S. Pat. No 5,445,961, thedisclosures of which are specifically incorporated herein by reference.

[0185] Although not required, when tassels are employed as the plantorgan, it is generally preferred to sterilize their surface. Followingsurface sterilization of the tassels, for example, with a solution ofcalcium hypochloride, the anthers are removed from about 70 to 150spikelets (small portions of the tassels) and placed in a preculture orpretreatment medium. Larger or smaller amounts can be used depending onthe number of anthers.

[0186] When one elects to employ tassels directly, tassels arepreferably pretreated at a cold temperature for a predefined time,preferably at 10° C. for about 4 days. After pretreatment of a wholetassel at a cold temperature, dissected anthers are further pretreatedin an environment that diverts microspores from their developmentalpathway. The function of the preculture medium is to switch thedevelopmental program from one of pollen development to that ofembryoid/callus development. An embodiment of such an environment in theform of a preculture medium includes a sugar alcohol, for examplemannitol or sorbitol, inositol or the like. An exemplary synergisticcombination is the use of mannitol at a temperature of about 10° C. fora period ranging from about 10 to 14 days. In a preferred embodiment, 3ml of 0.3 M mannitol combined with 50 mg/l of ascorbic acid, silvernitrate, and colchicine is used for incubation of anthers at 10° C. forbetween 10 and 14 days. Another embodiment is to substitute sorbitol formannitol. The colchicine produces chromosome doubling at this earlystage. The chromosome doubling agent is preferably only present at thepreculture stage.

[0187] It is believed that the mannitol or other similar carbonstructure or environmental stress induces starvation and functions toforce microspores to focus their energies on entering developmentalstages. The cells are unable to use, for example, mannitol as a carbonsource at this stage. It is believed that these treatments confuse thecells causing them to develop as embryoids and plants from microspores.Dramatic increases in development from these haploid cells, as high as25 embryoids in 10⁴ microspores, have resulted from using these methods.

[0188] In embodiments where microspores are obtained from anthers,microspores can be released from the anthers into an isolation mediumfollowing the mannitol preculture step. One method of release is bydisruption of the anthers, for example, by chopping the anthers intopieces with a sharp instrument, such as a razor blade, scalpel, orWaring blender. The resulting mixture of released microspores, antherfragments, and isolation medium are then passed through a filter toseparate microspores from anther wall fragments. An embodiment of afilter is a mesh, more specifically, a nylon mesh of about 112 mm poresize. The filtrate which results from filtering themicrospore-containing solution is preferably relatively free of antherfragments, cell walls, and other debris.

[0189] In a preferred embodiment, isolation of microspores isaccomplished at a temperature below about 25° C. and preferably, at atemperature of less than about 15° C. Preferably, the isolation media,dispersing tool (e.g., razor blade), funnels, centrifuge tubes, anddispersing container (e.g., petri dish) are all maintained at thereduced temperature during isolation. The use of a precooled dispersingtool to isolate maize microspores has been reported (Gaillard et al.,1991).

[0190] Where appropriate and desired, the anther filtrate is then washedseveral times in isolation medium. The purpose of the washing andcentrifugation is to eliminate any toxic compounds which are containedin the non-microspore part of the filtrate and are created by thechopping process. The centrifugation is usually done at decreasing spinspeeds, for example, 1000, 750, and finally 500 rpms. The result of theforegoing steps is the preparation of a relatively pure tissue culturesuspension of microspores that are relatively free of debris and antherremnants.

[0191] To isolate microspores, an isolation media is preferred. Anisolation media is used to separate microspores from the anther wallswhile maintaining their viability and embryogenic potential. Anillustrative embodiment of an isolation media includes a 6% sucrose ormaltose solution combined with an antioxidant such as 50 mg/l ofascorbic acid, 0.1 mg/l biotin, and 400 mg/l of proline, combined with10 mg/l of nicotinic acid and 0.5 mg/l AgNO₃. In another embodiment, thebiotin and proline are omitted.

[0192] An isolation media preferably has a higher antioxidant levelwhere it is used to isolate microspores from a donor plant (a plant fromwhich a plant composition containing a microspore is obtained) that isfield grown in contrast to greenhouse grown. A preferred level ofascorbic acid in an isolation medium is from about 50 mg/l to about 125mg/l and, more preferably, from about 50 mg/l to about 100 mg/l.

[0193] One can find particular benefit in employing a support for themicrospores during culturing and subculturing. Any support thatmaintains the cells near the surface can be used. The microsporesuspension is layered onto a support, for example by pipetting. Thereare several types of supports which are suitable and are within thescope of the invention. An illustrative embodiment of a solid support isa TRANSWELL® culture dish. Another embodiment of a solid support fordevelopment of the microspores is a bilayer plate wherein liquid mediais on top of a solid base. Other embodiments include a mesh or amillipore filter. Preferably, a solid support is a nylon mesh in theshape of a raft. A raft is defined as an approximately circular supportmaterial which is capable of floating slightly above the bottom of atissue culture vessel, for example, a petri dish, of about a 60 or 100mm size, although any other laboratory tissue culture vessel willsuffice. In an illustrative embodiment, a raft is about 55 mm indiameter.

[0194] Culturing isolated microspores on a solid support, for example,on a 10 mm pore nylon raft floating on 2.2 ml of medium in a 60 mm petridish, prevents microspores from sinking into the liquid medium and thusavoiding low oxygen tension. These types of cell supports enable theserial transfer of the nylon raft with its associatedmicrospore/embryoids ultimately to full strength medium containingactivated charcoal and solidified with, for example, GELRITE™(solidifying agent). The charcoal is believed to absorb toxic wastes andintermediaries. The solid medium allows embryoids to mature.

[0195] The liquid medium passes through the mesh while the microsporesare retained and supported at the medium-air interface. The surfacetension of the liquid medium in the petri dish causes the raft to float.The liquid is able to pass through the mesh; consequently, themicrospores stay on top. The mesh remains on top of the total volume ofliquid medium. An advantage of the raft is to permit diffusion ofnutrients to the microspores. Use of a raft also permits transfer of themicrospores from dish to dish during subsequent subculture with minimalloss, disruption, or disturbance of the induced embryoids that aredeveloping. The rafts represent an advantage over the multi-welledTRANSWELL® plates, which are commercially available from COSTAR, in thatthe commercial plates are expensive. Another disadvantage of theseplates is that to achieve the serial transfer of microspores tosubsequent media, the membrane support with cells must be peeled off theinsert in the wells. This procedure does not produce as good a yield noras efficient transfers, as when a mesh is used as a vehicle for celltransfer.

[0196] The culture vessels can be further defined as either (1) abilayer 60 mm petri plate wherein the bottom 2 ml of medium aresolidified with 0.7% agarose overlaid with 1 mm of liquid containing themicrospores; (2) a nylon mesh raft wherein a wafer of nylon is floatedon 1.2 ml of medium and 1 ml of isolated microspores is pipetted on top;or (3) TRANSWELL® plates wherein isolated microspores are pipetted ontomembrane inserts which support the microspores at the surface of 2 ml ofmedium.

[0197] After the microspores have been isolated, they are cultured in alow strength anther culture medium until about the 50 cell stage whenthey are subcultured onto an embryoid/callus maturation medium. Mediumis defined at this stage as any combination of nutrients that permit themicrospores to develop into embryoids or callus. Many examples ofsuitable embryoid/callus promoting media are well known to those skilledin the art. These media will typically comprise mineral salts, a carbonsource, vitamins, and growth regulators. A solidifying agent isoptional. A preferred embodiment of such a media is referred to as “Dmedium,” which typically includes 6N1 salts, AgNO₃ and sucrose ormaltose.

[0198] In an illustrative embodiment, 1 ml of isolated microspores arepipetted onto a 10 mm nylon raft and the raft is floated on 1.2 ml ofmedium “D,” containing sucrose or preferably maltose. Both calli andembryoids can develop. Calli are undifferentiated aggregates of cells.Type I is a relatively compact, organized, and slow growing callus. TypeII is a soft, friable, and fast-growing one. Embryoids are aggregatesexhibiting some embryo-like structures. The embryoids are preferred forsubsequent steps to regenerating plants. Culture medium “D” is anembodiment of medium that follows the isolation medium and replaces it.Medium “D” promotes growth to an embryoid/callus. This medium comprises6N1 salts at ⅛ the strength of a basic stock solution (major components)and minor components, plus 12% sucrose, or preferably 12% maltose, 0.1mg/l Bi, 0.5 mg/l nicotinic acid, 400 mg/l proline and 0.5 mg/l silvernitrate. Silver nitrate is believed to act as an inhibitor to the actionof ethylene. Multi-cellular structures of approximately 50 cells eachgenerally arise during a period of 12 days to 3 weeks. Serial transferafter a two week incubation period is preferred.

[0199] After the petri dish has been incubated for an appropriate periodof time, preferably two weeks in the dark at a predefined temperature, araft bearing the dividing microspores is transferred serially to solidbased media which promote embryo maturation. In an illustrativeembodiment, the incubation temperature is 30° C. and the mesh raftsupporting the embryoids is transferred to a 100 mm petri dishcontaining the 6N1-TGR-4P medium, an “anther culture medium.” Thismedium contains 6N1 salts, supplemented with 0.1 mg/l TIBA, 12% sugar(sucrose, maltose, or a combination thereof), 0.5% activated charcoal,400 mg/l proline, 0.5 mg/l B, 0.5 mg/l nicotinic acid, and 0.2 percentGELRITE™ (solidifying agent) and is capable of promoting the maturationof the embryoids. Higher quality embryoids, that is, embryoids whichexhibit more organized development, such as better shoot meristemformation without precocious germination, were typically obtained withthe transfer to full strength medium compared to those resulting fromcontinuous culture using only, for example, the isolated microsporeculture (IMC) Medium “D.” The maturation process permits the pollenembryoids to develop further in route toward the eventual regenerationof plants. Serial transfer occurs to full strength solidified 6N1 mediumusing either the nylon raft, the TRANSWELL® membrane, or bilayer plates,each one requiring the movement of developing embryoids to permitfurther development into physiologically more mature structures. In anespecially preferred embodiment, microspores are isolated in anisolation media comprising about 6% maltose, cultured for about twoweeks in an embryoid/calli induction medium comprising about 12% maltoseand then transferred to a solid medium comprising about 12% sucrose.

[0200] At the point of transfer of the raft, after about two weeks ofincubation, embryoids exist on a nylon support. The purpose oftransferring the raft with the embryoids to a solidified medium afterthe incubation is to facilitate embryo maturation. Mature embryoids atthis point are selected by visual inspection indicated by zygoticembryo-like dimensions and structures and are transferred to the shootinitiation medium. It is preferred that shoots develop before roots, orthat shoots and roots develop concurrently. If roots develop beforeshoots, plant regeneration can be impaired. To produce solidified media,the bottom of a petri dish of approximately 100 mm is covered with about30 ml of 0.2% GELRITE™ solidified medium. A sequence of regenerationmedia are used for whole plant formation from the embryoids.

[0201] During the regeneration process, individual embryoids are inducedto form plantlets. The number of different media in the sequence canvary depending on the specific protocol used. Finally, a rooting mediumis used as a prelude to transplanting to soil. When plantlets reach aheight of about 5 cm, they are then transferred to pots for furthergrowth into flowering plants in a greenhouse by methods well known tothose skilled in the art.

[0202] Plants have been produced from isolated microspore cultures bythe methods disclosed herein, including self-pollinated plants. The rateof embryoid induction was much higher with the synergistic preculturetreatment consisting of a combination of stress factors, including acarbon source which can be capable of inducing starvation, a coldtemperature, and colchicine, than has previously been reported. Anillustrative embodiment of the synergistic combination of treatmentsleading to the dramatically improved response rate compared to priormethods, is a temperature of about 10° C., mannitol as a carbon source,and 0.05% colchicine.

[0203] The inclusion of ascorbic acid, an anti-oxidant, in the isolationmedium is preferred for maintaining good microspore viability. However,there seems to be no advantage to including mineral salts in theisolation medium. The osmotic potential of the isolation medium wasmaintained optimally with about 6% sucrose, although a range of 2% to12% is within the scope of this invention.

[0204] In an embodiment of the embryoid/callus organizing media, mineralsalts concentration in IMC Culture Media “D” is (⅛×), the concentrationwhich is used also in anther culture medium. The 6N1 salts majorcomponents have been modified to remove ammonium nitrogen. Osmoticpotential in the culture medium is maintained with about 12% sucrose andabout 400 mg/l proline. Silver nitrate (0.5 mg/l) was included in themedium to modify ethylene activity. The preculture media is furthercharacterized by having a pH of about 5.7 to 6.0. Silver nitrate andvitamins do not appear to be crucial to this medium but do improve theefficiency of the response.

[0205] Whole anther cultures can also be used in the production ofmonocotyledonous plants from a plant culture system. There are somebasic similarities of anther culture methods and microspore culturemethods with regard to the media used. A difference from isolatedmicrospore cultures is that undisrupted anthers are cultured, so that asupport, e.g., a nylon mesh support, is not needed. The first step indeveloping the anther cultures is to incubate tassels at a coldtemperature. A cold temperature is defined as less than about 25° C.More specifically, the incubation of the tassels is preferably performedat about 10° C. A range of 8 to 14° C. is also within the scope of theinvention. The anthers are then dissected from the tassels, preferablyafter surface sterilization using forceps, and placed on solidifiedmedium. An example of such a medium is designated 6N1-TGR-P4.

[0206] The anthers are then treated with environmental conditions thatare combinations of stresses that are capable of diverting microsporesfrom gametogenesis to embryogenesis. It is believed that the stresseffect of sugar alcohols in the preculture medium, for example,mannitol, is produced by inducing starvation at the predefinedtemperature. In one embodiment, the incubation pretreatment is for about14 days at 10° C. It was found that treating the anthers in additionwith a carbon structure, an illustrative embodiment being a sugaralcohol, preferably mannitol, produces dramatically higher antherculture response rates as measured by the number of eventuallyregenerated plants, than by treatment with either cold treatment ormannitol alone. These results are particularly surprising in light ofteachings that cold is better than mannitol for these purposes, and thatwarmer temperatures interact with mannitol better.

[0207] To incubate the anthers, they are floated on a preculture mediumwhich diverts the microspores from gametogenesis, preferably on amannitol carbon structure, more specifically, 0.3 M of mannitol plus 50mg/l of ascorbic acid. Three milliliters is about the total amount in adish, for example, a tissue culture dish, more specifically, a 60 mmpetri dish. Anthers are isolated from about 120 spikelets for one dishyields about 360 anthers.

[0208] Chromosome doubling agents can be used in the preculture mediafor anther cultures. Several techniques for doubling chromosome number(Jensen, 1974; Wan et al., 1989) have been described. Colchicine is oneof the doubling agents. However, developmental abnormalities arisingfrom in vitro cloning are further enhanced by colchicine treatments, andprevious reports indicated that colchicine is toxic to microspores. Theaddition of colchicine in increasing concentrations during mannitolpretreatment prior to anther culture and microspore culture has achievedimproved percentages.

[0209] An illustrative embodiment of the combination of a chromosomedoubling agent and preculture medium is one which contains colchicine.In a specific embodiment, the colchicine level is preferably about0.05%. The anthers remain in the mannitol preculture medium with theadditives for about 10 days at 10° C. Anthers are then placed onmaturation media, for example, that designated 6N1-TGR-P4, for 3 to 6weeks to induce embryoids. If the plants are to be regenerated from theembryoids, shoot regeneration medium is employed, as in the isolatedmicrospore procedure described in the previous sections. Otherregeneration media can be used sequentially to complete regeneration ofwhole plants.

[0210] The anthers are then exposed to embryoid/callus promoting medium,for example, that designated 6N1-TGR-P4, to obtain callus or embryoids.The embryoids are recognized visually by identification ofembryonic-like structures. At this stage, the embryoids are transferredprogressively through a series of regeneration media. In an illustrativeembodiment, the shoot initiation medium comprises BAP(6-benzyl-amino-purine) and NAA (naphthalene acetic acid). Regenerationprotocols for isolated microspore cultures and anther cultures aresimilar.

[0211] VIII. ADDITIONAL TISSUE CULTURES AND REGENERATION

[0212] The present invention contemplates a corn plant regenerated froma tissue culture of the inbred maize plant 3323, or of a hybrid maizeplant produced by crossing 3323. As is well known in the art, tissueculture of corn can be used for the in vitro regeneration of a cornplant. By way of example, a process of tissue culturing and regenerationof corn is described in European Patent Application 0 160 390, thedisclosure of which is incorporated herein by reference. Corn tissueculture procedures are also described in Green and Rhodes (1982) andDuncan et al. (1985). The study by Duncan et al. (1985) indicates that97 percent of cultured plants produced calli capable of regeneratingplants. Subsequent studies have shown that both inbreds and hybridsproduced 91% regenerable calli that produced plants.

[0213] Other studies indicate that non-traditional tissues are capableof producing somatic embryogenesis and plant regeneration (Songstad etal., 1988; Rao et al., 1986; Conger et al., 1987; the disclosures ofwhich are incorporated herein by reference). Regenerable cultures,including Type I and Type II cultures, may be initiated from immatureembryos using methods described in, for example, PCT Application WO95/06128, the disclosure of which is incorporated herein by reference inits entirety.

[0214] Briefly, by way of example, to regenerate a plant of thisinvention, cells are selected following growth in culture. Whereemployed, cultured cells are preferably grown either on solid supportsor in the form of liquid suspensions as set forth above. In eitherinstance, nutrients are provided to the cells in the form of media, andenvironmental conditions are controlled. There are many types of tissueculture media comprising amino acids, salts, sugars, hormones, andvitamins. Most of the media employed to regenerate inbred and hybridplants have some similar components; the media differ in the compositionand proportions of their ingredients depending on the particularapplication envisioned. For example, various cell types usually grow inmore than one type of media, but exhibit different growth rates anddifferent morphologies, depending on the growth media. In some media,cells survive but do not divide. Various types of media suitable forculture of plant cells have been previously described and discussedabove.

[0215] An exemplary embodiment for culturing recipient corn cells insuspension cultures includes using embryogenic cells in Type II(Armstrong and Green, 1985; Gordon-Kamm et al., 1990) callus, selectingfor small (10 to 30 mm) isodiametric, cytoplasmically dense cells,growing the cells in suspension cultures with hormone containing media,subculturing into a progression of media to facilitate development ofshoots and roots, and finally, hardening the plant and readying itmetabolically for growth in soil.

[0216] Meristematic cells (i.e., plant cells capable of continual celldivision and characterized by an undifferentiated cytologicalappearance, normally found at growing points or tissues in plants suchas root tips, stem apices, lateral buds, etc.) can be cultured (U.S.Pat. No 5,736,369, the disclosure of which is specifically incorporatedherein by reference).

[0217] Embryogenic calli are produced essentially as described in PCTApplication WO 95/06128. Specifically, inbred plants or plants fromhybrids produced from crossing an inbred of the present invention withanother inbred are grown to flowering in a greenhouse. Explants from atleast one of the following F₁ tissues: the immature tassel tissue,intercalary meristems and leaf bases, apical meristems, immature earsand immature embryos are placed in an initiation medium which contain MSsalts, supplemented with thiamine, agar, and sucrose. Cultures areincubated in the dark at about 23° C. All culture manipulations andselections are performed with the aid of a dissecting microscope.

[0218] After about 5 to 7 days, cellular outgrowths are observed fromthe surface of the explants. After about 7 to 21 days, the outgrowthsare subcultured by placing them into fresh medium of the samecomposition. Some of the intact immature embryo explants are placed onfresh medium. Several subcultures later (after about 2 to 3 months)enough material is present from explants for subdivision of theseembryogenic calli into two or more pieces.

[0219] Callus pieces from different explants are not mixed. Afterfurther growth and subculture (about 6 months after embryogenic callusinitiation), there are usually between I and 100 pieces derivedultimately from each selected explant. During this time of cultureexpansion, a characteristic embryogenic culture morphology develops as aresult of careful selection at each subculture. Any organized structuresresembling roots or root primordia are discarded. Material known fromexperience to lack the capacity for sustained growth is also discarded(translucent, watery, embryogenic structures). Structures with a firmconsistency resembling at least in part the scutelum of the in vivoembryo are selected.

[0220] The callus is maintained on agar-solidified MS or N6-type media.A preferred hormone is 2,4-D. A second preferred hormone is dicamba.Visual selection of embryo-like structures is done to obtainsubcultures. Transfer of material other than that displaying embryogenicmorphology results in loss of the ability to recover whole plants fromthe callus.

[0221] Cell suspensions are prepared from the calli by selecting cellpopulations that appear homogeneous macroscopically. A portion of thefriable, rapidly growing embryogenic calli is inoculated into MS or N6Medium containing 2,4-D or dicamba. The calli in medium are incubated atabout 27° C. on a gyrotary shaker in the dark or in the presence of lowlight. The resultant suspension culture is transferred about once everythree to seven days, preferably every three to four days, by takingabout 5 to 10 ml of the culture and introducing this inoculum into freshmedium of the composition listed above (PCT Application WO 95/06128).

[0222] For regeneration of type I or type II callus, callus istransferred to a solidified culture medium which includes a lowerconcentration of 2,4-D or other auxins than is present in culture mediumused for callus maintenance (PCT Application WO 95/06128, specificallyincorporated herein by reference). Other hormones which can be used inregeneration media include dicamba, NAA, ABA, BAP, and 2-NCA.Regeneration of plants is completed by the transfer of mature andgerminating embryos to a hormone-free medium, followed by the transferof developed plantlets to soil and growth to maturity. Plantregeneration is described in PCT Application WO 95/06128.

[0223] Cells from the meristem or cells fated to contribute to themeristem of a cereal plant embryo at the early proembryo, mid proembryo,late proembryo, transitional or early coleoptilar stage may be culturedso as to produce a proliferation of shoots or multiple meristems fromwhich fertile plants may be regenerated. Alternatively, cells from themeristem or cells fated to contribute to the meristem of a cereal plantimmature ear or tassel may be cultured so as to produce a proliferationof shoots or multiple meristems from which fertile plants may beregenerated (U.S. Pat. No. 5,736,369).

[0224] Progeny of any generation are produced by taking pollen andselfing, backcrossing, or sibling crossing regenerated plants by methodswell known to those skilled in the arts. Seeds are collected from theregenerated plants. Alternatively, progeny of any generation may beproduced by pollinating a regenerated plant with its own pollen orpollen of a second maize plant. Using the methods described herein,tissue cultures and immature or mature plant tissues may be used asrecipient cell cultures for the process of genetic transformation.

[0225] IX. PROCESSES OF PREPARING CORN PLANTS AND THE CORN PLANTSPRODUCED BY SUCH CROSSES

[0226] The present invention also provides a process of preparing anovel corn plant and a corn plant produced by such a process. Inaccordance with such a process, a first parent corn plant is crossedwith a second parent corn plant wherein at least one of the first andsecond corn plants is the inbred corn plant 3323. An important aspect ofthis process is that it can be used for the development of novel inbredlines. For example, the inbred corn plant 3323 could be crossed to anysecond plant, and the resulting hybrid progeny each selfed for about 5to 7 or more generations, thereby providing a large number of distinct,pure-breeding inbred lines. These inbred lines could then be crossedwith other inbred or non-inbred lines and the resulting hybrid progenyanalyzed for beneficial characteristics. In this way, novel inbred linesconferring desirable characteristics could be identified.

[0227] In selecting a second plant to cross with 3323 for the purpose ofdeveloping novel inbred lines, it will typically be desired choose thoseplants which either themselves exhibit one or more selected desirablecharacteristics or which exhibit the desired characteristic(s) when inhybrid combination. Examples of potentially desired characteristicsinclude greater yield, better stalks, better roots, resistance toinsecticides, herbicides, pests, and disease, tolerance to heat anddrought, reduced time to crop maturity, better agronomic quality, highernutritional value, and uniformity in germination times, standestablishment, growth rate, maturity, and fruit size. Alternatively, theinbred 3323 may be crossed with a second, different inbred plant for thepurpose of producing hybrid seed which is sold to farmers for plantingin commercial production fields. In this case, a second inbred varietyis selected which confers desirable characteristics when in hybridcombination with the first inbred line.

[0228] Corn plants (Zea mays L.) can be crossed by either natural ormechanical techniques. Natural pollination occurs in corn when windblows pollen from the tassels to the silks that protrude from the topsof the recipient ears. Mechanical pollination can be effected either bycontrolling the types of pollen that can blow onto the silks or bypollinating by hand.

[0229] In a preferred embodiment, crossing comprises the steps of:

[0230] (a) planting in pollinating proximity seeds of a first and asecond parent corn plant, and preferably, seeds of a first inbred cornplant and a second, distinct inbred corn plant;

[0231] (b) cultivating or growing the seeds of the first and secondparent corn plants into plants that bear flowers;

[0232] (c) emasculating flowers of either the first or second parentcorn plant, i.e., treating the flowers so as to prevent pollenproduction, or alternatively, using as the female parent a male sterileplant, thereby providing an emasculated parent corn plant;

[0233] (d) allowing natural cross-pollination to occur between the firstand second parent corn plants;

[0234] (e) harvesting seeds produced on the emasculated parent cornplant; and, where desired,

[0235] (f) growing the harvested seed into a corn plant, preferably, ahybrid corn plant.

[0236] Parental plants are typically planted in pollinating proximity toeach other by planting the parental plants in alternating rows, inblocks or in any other convenient planting pattern. Where the parentalplants differ in timing of sexual maturity, it may be desired to plantthe slower maturing plant first, thereby ensuring the availability ofpollen from the male parent during the time at which silks on the femaleparent are receptive to pollen. Plants of both parental parents arecultivated and allowed to grow until the time of flowering.Advantageously, during this growth stage, plants are in general treatedwith fertilizer and/or other agricultural chemicals as consideredappropriate by the grower.

[0237] At the time of flowering, in the event that plant 3323 isemployed as the male parent, the tassels of the other parental plant areremoved from all plants employed as the female parental plant to avoidself-pollination. The detasseling can be achieved manually but also canbe done by machine, if desired. Alternatively, when the female parentcorn plant comprises a cytoplasmic or nuclear gene conferring malesterility, detasseling may not be required. Additionally, a chemicalgametocide may be used to sterilize the male flowers of the femaleplant. In this case, the parent plants used as the male may either notbe treated with the chemical agent or may comprise a genetic factorwhich causes resistance to the emasculating effects of the chemicalagent. Gametocides affect processes or cells involved in thedevelopment, maturation or release of pollen. Plants treated with suchgametocides are rendered male sterile, but typically remain femalefertile. The use of chemical gametocides is described, for example, inU.S. Pat. No. 4,936,904, the disclosure of which is specificallyincorporated herein by reference in its entirety. Furthermore, the useof Roundup herbicide in combination with glyphosate tolerant maizeplants to produce male sterile corn plants is disclosed in U.S. patentapplication Ser. No. 08/927,368 and PCT Publication WO 98/44140.

[0238] Following emasculation, the plants are then typically allowed tocontinue to grow and natural cross-pollination occurs as a result of theaction of wind, which is normal in the pollination of grasses, includingcorn. As a result of the emasculation of the female parent plant, allthe pollen from the male parent plant is available for pollinationbecause tassels, and thereby pollen bearing flowering parts, have beenpreviously removed from all plants of the inbred plant being used as thefemale in the hybridization. Of course, during this hybridizationprocedure, the parental varieties are grown such that they are isolatedfrom other corn fields to minimize or prevent any accidentalcontamination of pollen from foreign sources. These isolation techniquesare well within the skill of those skilled in this art.

[0239] Both parental inbred plants of corn may be allowed to continue togrow until maturity or the male rows may be destroyed after flowering iscomplete. Only the ears from the female inbred parental plants areharvested to obtain seeds of a novel F₁ hybrid. The novel F₁ hybrid seedproduced can then be planted in a subsequent growing season incommercial fields or, alternatively, advanced in breeding protocols forpurposes of developing novel inbred lines.

[0240] Alternatively, in another embodiment of the invention, both firstand second parent corn plants can come from the same inbred corn plant,i.e., from the inbred designated 3323. Thus, any corn plant producedusing a process of the present invention and inbred corn plant 3323, iscontemplated by the current inventor. As used herein, crossing can meanselfing, backcrossing, crossing to another or the same inbred, crossingto populations, and the like. All corn plants produced using the inbredcorn plant 3323 as a parent are, therefore, within the scope of thisinvention.

[0241] The utility of the inbred plant 3323 also extends to crosses withother species. Commonly, suitable species will be of the familyGraminaceae, and especially of the genera Zea, Tripsacum, Coix,Schlerachne, Polytoca, Chionachne, and Trilobachne, of the tribeMaydeae. Of these, Zea and Tripsacum, are most preferred. Potentiallysuitable for crosses with 3323 can also be the various varieties ofgrain sorghum, Sorghum bicolor (L.) Moench.

[0242] A. F₁ Hybrid Corn Plant and Seed Production

[0243] Any time the inbred corn plant 3323 is crossed with another,different, corn inbred, a first generation (F₁) corn hybrid plant isproduced. As such, an F₁ hybrid corn plant may be produced by crossing3323 with any second inbred maize plant. Therefore, any F₁ hybrid cornplant or corn seed which is produced with 3323 as a parent is part ofthe present invention. An example of such an F₁ hybrid which has beenproduced with 3323 as a parent is the hybrid 0993357.

[0244] The goal of the process of producing an F₁ hybrid is tomanipulate the genetic complement of corn to generate new combinationsof genes which interact to yield new or improved traits (phenotypiccharacteristics). A process of producing an F₁ hybrid typically beginswith the production of one or more inbred plants. Those plants areproduced by repeated crossing of ancestrally related corn plants to tryto combine certain genes within the inbred plants.

[0245] Corn has a diploid phase which means two conditions of a gene(two alleles) occupy each locus (position on a chromosome). If thealleles are the same at a locus, there is said to be homozygosity. Ifthey are different, there is said to be heterozygosity. In a completelyinbred plant, all loci are homozygous. Because many loci when homozygousare deleterious to the plant, in particular leading to reduced vigor,less kernels, weak and/or poor growth, production of inbred plants is anunpredictable and arduous process. Under some conditions, heterozygousadvantage at some loci effectively bars perpetuation of homozygosity.

[0246] Inbreeding requires sophisticated manipulation by human breeders.Even in the extremely unlikely event inbreeding rather thancrossbreeding occurred in natural corn, achievement of completeinbreeding cannot be expected in nature due to well known deleteriouseffects of homozygosity and the large number of generations the plantwould have to breed in isolation. The reason for the breeder to createinbred plants is to have a known reservoir of genes whose gametictransmission is predictable.

[0247] The development of inbred plants generally requires at leastabout 5 to 7 generations of selfing. Inbred plants are then cross-bredin an attempt to develop improved F₁ hybrids. Hybrids are then screenedand evaluated in small scale field trials. Typically, about 10 to 15phenotypic traits, selected for their potential commercial value, aremeasured. A selection index of the most commercially important traits isused to help evaluate hybrids. FACT, an acronym for Field AnalysisComparison Trial (strip trials), is an on-farm experimental testingprogram employed by DEKALB Genetics Corporation to perform the finalevaluation of the commercial potential of a product.

[0248] During the next several years, a progressive elimination ofhybrids occurs based on more detailed evaluation of their phenotype.Eventually, strip trials (FACT) are conducted to formally compare theexperimental hybrids being developed with other hybrids, some of whichwere previously developed and generally are commercially successful.That is, comparisons of experimental hybrids are made to competitivehybrids to determine if there was any advantage to further developmentof the experimental hybrids. Examples of such comparisons are presentedhereinbelow. After FACT testing is complete, determinations may be madewhether commercial development should proceed for a given hybrid.

[0249] When the inbred corn plant 3323 is crossed with another inbredplant to yield a hybrid, the original inbred can serve as either thematernal or paternal plant. For many crosses, the outcome is the sameregardless of the assigned sex of the parental plants. However, there isoften one of the parental plants that is preferred as the maternal plantbecause of increased seed yield and production characteristics. Someplants produce tighter ear husks leading to more loss, for example dueto rot. There can be delays in silk formation which deleteriously affecttiming of the reproductive cycle for a pair of parental inbreds. Seedcoat characteristics can be preferable in one plant. Pollen can be shedbetter by one plant. Other variables can also affect preferred sexualassignment of a particular cross. In the case of the instant inbred, itwas generally preferable to use 3323 as the male parent.

[0250] B. F₁ Hybrid Comparisons

[0251] As mentioned above, hybrids are progressively eliminatedfollowing detailed evaluations of their phenotype, including formalcomparisons with other commercially successful hybrids. Strip trials areused to compare the phenotypes of hybrids grown in as many environmentsas possible. They are performed in many environments to assess overallperformance of the new hybrids and to select optimum growing conditions.Because the corn is grown in close proximity, environmental factors thataffect gene expression, such as moisture, temperature, sunlight, andpests, are minimized. For a decision to be made to commercialize ahybrid, it is not necessary that the hybrid be better than all otherhybrids. Rather, significant improvements must be shown in at least sometraits that would create improvements in some niches.

[0252] Examples of such comparative data are set forth herein below inTable 4, which presents a comparison of performance data for the hybrid0993357, a hybrid made with 3323 as one parent, versus selected hybridsof commercial value (DK655 and DK658). All the data in Table 4represents results across years and locations for research and/or striptrials. The “NTEST” represents the number of paired observations indesignated tests at locations around the United States. TABLE 4Comparative Data of 0993357 SI YLD MST STL RTL DRP FLSTD SV ELSTD PHTEHT BAR SG TST ESTR HYBRID NTEST % C BU PTS % % % % M RAT % M INCH INCH% RAT LBS FGDU DAYS 0993357 R 10 91.2 170.7 21.9 3.6 0.0 96.4 97.7 43.755.3 113.7 DK655 100.7 183.8 22.4 0.7 0.0 98.6 95.7 44.7 56.7 114.4 DIFF−9.6 −13.1 −0.5 3.0 0.0 −2.2 2.0 −1.0 −1.4 −0.7 SIG * ** + + 0993357 F46 94.6 176.2 19.4 1.9 0.7 0.0 91.2 4.0 90.5 87.3 41.0 0.0 3.2 57.1 1255113.6 DK655 101.4 185.4 19.5 6.2 0.7 0.0 102.7 2.5 100.4 89.0 44.3 8.25.8 58.7 1335 113.4 DIFF −6.8 −9.2 −0.1 −4.3 0.0 0.0 −11.5 1.5 −9.8 −1.8−3.3 −8.2 −2.6 −1.6 −80 0.3 SIG * * ** + ** ** 0993357 F 74 99.0 168.120.1 2.0 0.2 0.0 93.7 4.6 94.8 92.6 42.2 3.1 3.7 54.8 1318 113.8 DK65896.7 169.1 20.7 2.9 5.2 0.1 100.8 3.8 98.7 98.2 47.2 4.6 3.9 55.4 1399114.3 DIFF 2.3 −1.0 −0.5 −0.9 −5.0 −0.1 −7.1 0.8 −3.9 −5.7 −4.9 −1.5−0.2 −0.6 −81 −0.5 SIG * ** * + ** ** ** ** +

[0253] As can be seen in Table 4, the hybrid 0993357 differssignificantly in several aspects when compared to the successfulcommercial hybrids DK655 and DK658.

[0254] C. Physical Description of F₁ Hybrids

[0255] The present invention provides F₁ hybrid corn plants derived fromthe corn plant 3323. Examples of physical characteristics of anexemplary hybrid produced using 3323 are listed below, in Table 5. TABLE5 Morphological Traits for the 0993357 Phenotype CHARACTERISTIC VALUE 1.STALK Diameter (width) cm.  2.2 Anthocyanin Absent Nodes With BraceRoots  1.7 Brace Root Color Faint Internode Direction Straight InternodeLength cm. 23.3 2. LEAF Color Green Length cm. 87.9 Width cm.  9.5Sheath Anthocyanin Basal-Weak Sheath Pubescence Moderate Marginal WavesModerate Longitudinal Creases Moderate 3. TASSEL Attitude Upright Lengthcm. 51.9 Spike Length cm. 23.9 Peduncle Length cm. 17.7 Branch Number 7.6 Anther Color Yellow Glume Color Green Glume Band Absent 4. EAR SilkColor Yellow Number Per Stalk  1.0 Position (attitude) Upright Lengthcm. 16.5 Shape Semi-Conical Diameter cm.  5.2 Shank Length cm.  9.3 HuskBract Short Husk Cover cm.  4.2 Husk Opening Intermediate Husk ColorFresh Green Husk Color Dry Buff Cob Diameter cm.  5.2 Cob Color RedShelling Percent 87.6 5. KERNEL Row Number 18.6 Number Per Row 39   RowDirection Slightly Curved Type Dent Cap Color Yellow Side Color DeepYellow Length (depth) mm. 13.8 Width mm.  7.5 Thickness  4.1 EndospermType Normal Endo sperm Color Pale-Yellow

[0256] X. GENETIC COMPLEMENTS

[0257] The present invention provides a genetic complement of the inbredcorn plant designated 3323. Further provided by the invention is ahybrid genetic complement, wherein the complement is formed by thecombination of a haploid genetic complement from 3323 and anotherhaploid genetic complement. Means for determining such a geneticcomplement are well-known in the art.

[0258] As used herein, the phrase “genetic complement” means anaggregate of nucleotide sequences, the expression of which defines thephenotype of a corn plant or a cell or tissue of that plant. By way ofexample, a corn plant is genotyped to determine a representative sampleof the inherited markers it possesses. Markers are alleles at a singlelocus. They are preferably inherited in codominant fashion so that thepresence of both alleles at a diploid locus is readily detectable, andthey are free of environmental variation, i.e., their heritability is 1.This genotyping is preferably performed on at least one generation ofthe descendant plant for which the numerical value of the quantitativetrait or traits of interest are also determined. The array of singlelocus genotypes is expressed as a profile of marker alleles, two at eachlocus. The marker allelic composition of each locus can be eitherhomozygous or heterozygous. Homozygosity is a condition where bothalleles at a locus are characterized by the same nucleotide sequence orsize of a repeated sequence. Heterozygosity refers to differentconditions of the gene at a locus. A preferred type of genetic markerfor use with the invention is simple sequence repeats (SSRs), althoughpotentially any other type of genetic marker could be used, for example,restriction fragment length polymorphisms (RFLPs), amplified fragmentlength polymorphisms (AFLPs), single nucleotide polymorphisms (SNPs),and isozymes.

[0259] A genetic marker profile of an inbred may be predictive of theagronomic traits of a hybrid produced using that inbred. For example, ifan inbred of known genetic marker profile and phenotype is crossed witha second inbred of known genetic marker profile and phenotype it ispossible to predict the phenotype of the F₁ hybrid based on the combinedgenetic marker profiles of the parent inbreds. Methods for prediction ofhybrid performance from genetic marker data is disclosed in U.S. Pat.No. 5,492,547, the disclosure of which is specifically incorporatedherein by reference in its entirety. Such predictions may be made usingany suitable genetic marker, for example, SSRs, RFLPs, AFLPs, SNPs, orisozymes.

[0260] SSRs are genetic markers based on polymorphisms in repeatednucleotide sequences, such as microsatellites. A marker system based onSSRs can be highly informative in linkage analysis relative to othermarker systems in that multiple alleles may be present. Anotheradvantage of this type of marker is that, through use of flankingprimers, detection of SSRs can be achieved, for example, by thepolymerase chain reaction (PCRTm), thereby eliminating the need forlabor-intensive Southern hybridization. The PCRTM detection is done byuse of two oligonucleotide primers flanking the polymorphic segment ofrepetitive DNA. Repeated cycles of heat denaturation of the DNA followedby annealing of the primers to their complementary sequences at lowtemperatures, and extension of the annealed primers with DNA polymerase,comprise the major part of the methodology. Following amplification,markers can be scored by gel electrophoresis of the amplificationproducts. Scoring of marker genotype is based on the size (number ofbase pairs) of the amplified segment.

[0261] Means for performing genetic analyses using SSR polymorphisms arewell known in the art. The SSR analyses reported herein were conductedby Celera AgGen in Davis, Calif. This service is available to the publicon a contractual basis. This analysis was carried out by amplificationof simple repeats followed by detection of marker genotypes using gelelectrophoresis. Markers were scored based on the size of the amplifiedfragment.

[0262] The SSR genetic marker profile of the parental inbreds andexemplary resultant hybrid described herein were determined. Because aninbred is essentially homozygous at all relevant loci, an inbred should,in almost all cases, have only one allele at each locus. In contrast, adiploid genetic marker profile of a hybrid should be the sum of thoseparents, e.g., if one inbred parent had the allele 168 (base pairs) at aparticular locus, and the other inbred parent had 172, the hybrid is168.172 by inference. Subsequent generations of progeny produced byselection and breeding are expected to be of genotype 168, 172, or168.172 for that locus position. When the F₁ plant is used to produce aninbred, the locus should be either 168 or 172 for that position.Surprisingly, it has been observed that in certain instances, novel SSRgenotypes arise during the breeding process. An SSR genetic markerprofile of 3323 and two comparative inbreds is presented in Table 6.TABLE 6 SSR Profile of 3323 and Comparative Inbreds LOCUS 3323 3327 5750BNGL105  82  82  82 BNGL118 119 119 127 BNGL149 193 193 193 BNGL244 145— 186 BNGL252 168 164 168 BNGL426 119 127 119 BNGL589 157 175 157BNGL615 194 194 194 BNGL619 237 237 237 DUP14  76  76  76 DUP28  99  99147 MC1017 200 200 200 MC1018 136 136 136 MC1028 151 148 151 MC1043 159159 165 MC1074 186 180 164 MC1079 175 175 173 MC1094 172 170 172 MC1108142 144 144 MC1129 204 204 204 MC1131 111 111 117 MC1138 186 186 190MC1176 220 220 220 MC1189 220 222 220 MC1191 219 219 211 MC1194 143 —143 MC1208 115 115 111 MC1237 159 159 159 MC1257 187 187 180 MC1265 218244 218 MC1287 160 160 158 MC1288 113 113 113 MC1325 153 153 185 MC1329111 —  93 MC1360 145 149 149 MC1371 124 132 124 MC1449 148 148  97MC1456 195 195 191 MC1484 124 124 124 MC1520 283 300 283 MC1523 201 201200 MC1526 114 114 124 MC1538 223 223 237 MC1605 110 110 110 MC1662 134167 136 MC1720 239 239 227 MC1732 108 108 108 MC1740 129 157 140 MC1782228 228 265 MC1784 252 250 252 MC1808 147 131 147 MC1831 182 182 196MC1834 208 208 208 MC1839 188 196 196 MC1866 123 123 123 MC1890 142 —142 MC1904 172 172 191 MC2047 144 144 144 MC2086 240 240 247 MC2122 220236 236 MC2132 223 223 223 MC2238 195 195 182 MC2259 177 177 177 MC2305180 180 190 NC004 156 156 156 NC009 134 134 134 PHI017 110 110 110PHI031 198 198 198 PHI033 257 257 257 PHI037 161 161 160 PHI050  86  86 92 PHI051 149 149 149 PHI061  93  93  93 PHI064  84  84  84 PHI065 158158 158 PHI078 129 129 129 PHI096 109 109 109 PHI116 181 181 160 PHI119168 174 168 PHI120  76  76  76

[0263] Another aspect of this invention is a plant genetic complementcharacterized by a genetic isozyme typing profile. Isozymes are forms ofproteins that are distinguishable, for example, on starch gelelectrophoresis, usually by charge and/or molecular weight. Thetechniques and nomenclature for isozyme analysis are described in, forexample, Stuber et al. (1988), which is incorporated by reference.

[0264] A standard set of loci can be used as a reference set.Comparative analysis of these loci is used to compare the purity ofhybrid seeds, to assess the increased variability in hybrids compared toinbreds, and to determine the identity of seeds, plants, and plantparts. In this respect, an isozyme reference set can be used to developgenotypic “fingerprints.”

[0265] Table 7 lists the identifying numbers of the alleles at isozymeloci types, and represents the exemplary genetic isozyme typing profilefor 3323. TABLE 7 Isozyme Profile of 3323 and Comparative InbredsISOZYME ALLELE LOCI 3323 3327 5750 Acph1 4 3 4 Idh1 4 4 4 Idh2 4 4 6Mdh1 6 6 6 Mdh2 6 6 3.5 Mdh3 16 16 16 Mdh4 12 12 12 Mdh5 12 12 12 Pgm1 99 9 Pgm2 4 4 4 6Pgd1 3.8 3.8 3.8 6Pgd2 5 5 5

[0266] The present invention also provides a hybrid genetic complementformed by the combination of a haploid genetic complement of the cornplant 3323 with a haploid genetic complement of a second corn plant.Means for combining a haploid genetic complement from the foregoinginbred with another haploid genetic complement can comprise any methodfor producing a hybrid plant from 3323. It is contemplated that such ahybrid genetic complement can be prepared using in vitro regeneration ofa tissue culture of a hybrid plant of this invention.

[0267] A hybrid genetic complement contained in the seed of a hybridderived from 3323 is a further aspect of this invention. An exemplaryhybrid genetic complement is that of the hybrid 0993357.

[0268] Table 8 shows the identifying numbers of the alleles for thehybrid 0993357, which constitutes an exemplary SSR genetic markerprofile for hybrids derived from the inbred of the present invention.Table 8 concerns 0993357, which has 3323 as one inbred parent. TABLE 8SSR Profile of 0993357 PROBE Hybrid 0993357 BNGL105 82.94 BNGL118119.110 BNGL244 145.145 BNGL252 168.164 BNGL426 119.119 BNGL589 157.175BNGL615 194.231 BNGL619 237.265 DUP14  76.112 DUP28  99.131 MC1017200.196 MC1018 136.164 MC1028 151.167 MC1074 186.180 MC1079 175.173MC1094 172.184 MC1108 142.144 MC1129 204.206 MC1131 111.109 MC1138186.186 MC1176 220.220 MC1189 220.219 MC1191 219.206 MC1194 143.143MC1208 115.127 MC1237 159.161 MC1257 187.229 MC1287 160.156 MC1288113.113 MC1325 153.177 MC1329 111.93  MC1360 145.145 MC1449 148.160MC1456 195.190 MC1484 124.124 MC1523 201.199 MC1526 114.124 MC1538223.237 MC1605 110.128 MC1662 134.161 MC1720 239.241 MC1732 108.108MC1740 129.120 MC1782 228.228 MC1784 252.250 MC1808 147.137 MC1831182.186 MC1834 208.216 MC1866 123.119 MC1890 142.136 MC1904 172.193MC2047 144.148 MC2086 240.242 MC2122 220.254 MC2132 223.254 MC2259177.203 MC2305 180.218 NC004 156.156 NC009 134.119 PHI017 110.110 PHI031198.231 PHI033 257.257 PHI037 161.141 PHI050 86.92 PHI051 149.149 PHI06193.93 PHI064  84.104 PHI065 158.158 PHI078 129.129 PHI093 292.299 PHI116181.177 PHI119 168.174 PHI120 76.76

[0269] The exemplary hybrid genetic complements of hybrid 0993357 mayalso be assessed by genetic isozyme typing profiles using a standard setof loci as a reference set, using, e.g., the same, or a different, setof loci to those described above. Table 9 lists the identifying numbersof the alleles at isozyme loci types and presents the exemplary geneticisozyme typing profile for the hybrid 0993357, which is an exemplaryhybrid derived from the inbred of the present invention. Table 9concerns 0993357, which has 3323 as one inbred parent. TABLE 9 IsozymeProfile for Hybrid 0993357 Loci Isozyme Allele Acph1 4/2 Idh1 4 Idh2 4Mdh1 6 Mdh2   6/3.5 Mdh3 16 Mdh4 12 Mdh5 12 Pgm1 9 Pgm2 4 6-Pgd1 3.86-Pgd2 5

[0270] All of the compositions and methods disclosed and claimed hereincan be made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of the foregoing illustrative embodiments,it will be apparent to those of skill in the art that variations,changes, modifications, and alterations may be applied to thecomposition, methods, and in the steps or in the sequence of steps ofthe methods described herein, without departing from the true concept,spirit, and scope of the invention. More specifically, it will beapparent that certain agents that are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope, and concept of theinvention as defined by the appended claims.

REFERENCES

[0271] The following references, to the extent that they provideexemplary procedural or other details supplementary to those set forthherein, are specifically incorporated herein by reference.

[0272] Anderson, W. P., Weed Science Principles, West PublishingCompany, 1983.

[0273] Armstrong and Green, “Establishment and maintenance of friable,embryogenic maize callus and the involvement of L-proline,” Planta,164:207-214, 1985.

[0274] Conger, Novak, Afza, Erdelsky, “Somatic Embryogenesis fromCultured Leaf Segments of Zea Mays,” Plant Cell Reports, 6:345-347,1987.

[0275] Duncan et al., “The Production of Callus Capable of PlantRegeneration from Immature Embryos of Numerous ZeaMays Genotypes,”Planta, 165:322-332, 1985.

[0276] Fehr, “Theory and Technique,” In: Principles of CultivarDevelopment, 1:360-376, 1987.

[0277] Gaillard et al., “Optimization of Maize Microspore Isolation andCulture Condition for Reliable Plant Regeneration,” Plant Cell Reports,10(2): 55, 1991.

[0278] Gordon-Kamm et al., “Transformation of Maize Cells andRegeneration of Fertile Transgenic Plants,” The Plant Cell, 2:603-618,1990.

[0279] Green and Rhodes, “Plant Regeneration in Tissue Cultures ofMaize: Callus Formation from Stem Protoplasts of Corn (Zea Mays L.),”In: Maize for Biological Research, 367-372, 1982.

[0280] Jensen, “Chromosome Doubling Techniques in Haploids,” Haploidsand Higher Plants—Advances and Potentials, Proceedings of the FirstInternational Symposium, 1974.

[0281] Nienhuis et al., “Restriction Fragment Length PolymorphismAnalysis of Loci Associated with Insect Resistance in Tomato,” CropScience, 27(4): 797-803, 1987.

[0282] Omirulleh et al., “Activity of a chimeric promoter with thedoubled CaMV 35S enhancer element in protoplast-derived cells andtransgenic plants in maize,” Plant Mol. Biol., 21(3): 415-428, 1993.

[0283] Pace et al., “Anther Culture of Maize and the Visualization ofEmbryogenic Microspores by Fluorescent Microscopy,” Theoretical andApplied Genetics, 73:863-869, 1987.

[0284] Poehlman et aL, “Breeding Field Crops,” 4th Ed., Iowa StateUniversity Press, Ames, Iowa, pp 132-155 and 321-344, 1995.

[0285] Rao et al., “Somatic Embryogenesis in Glume Callus Cultures,”Maize Genetics Cooperation Newsletter, 60, 1986.

[0286] Songstad et al., “Effect of 1-Aminocyclopropate-1-CarboxylicAcid, Silver Nitrate, and Norbomadiene on Plant Regeneration from MaizeCallus Cultures,” Plant Cell Reports, 7:262-265, 1988.

[0287] Sprague and Dudley (eds.), “Corn and Corn Improvement,” 3rd Ed.,Crop Science of America, Inc., and Soil Science of America, Inc.,Madison Wis. pp 881-883 and pp 901-918, 1988.

[0288] Stuber et al., “Techniques and scoring procedures for starch gelelectrophoresis of enzymes of maize C. Zea mays, L.,” Tech. Bull., 286,1988.

[0289] Wan et al., “Efficient Production of Doubled Haploid PlantsThrough Colchicine Treatment of Anther-Derived Maize Callus,”Theoretical and Applied Genetics, 77:889-892, 1989.

[0290] Wang et al., “Large-Scale Identification, Mapping, and Genotypingof Single-Nucleotide Polymorphisms in the Human Genome,” Science,280:1077-1082, 1998.

[0291] Williams et al., “Oligonucleotide Primers of Arbitrary SequenceAmplify DNA Polymorphisms which Are Useful as Genetic Markers,” NucleicAcids Res., 18:6531-6535, 1990.

What is claimed is:
 1. Inbred corn seed of the corn plant 3323, a sampleof said seed having been deposited under ATCC Accession No. ______. 2.The inbred corn seed of claim 1, further defined as an essentiallyhomogeneous population of inbred corn seed.
 3. The inbred corn seed ofclaim 1, further defined as essentially free from hybrid seed.
 4. Aninbred corn plant produced by growing the seed of the inbred corn plant3323, a sample of said seed having been deposited under ATCC AccessionNo. ______.
 5. Pollen of the plant of claim
 4. 6. An ovule of the plantof claim
 4. 7. An essentially homogeneous population of corn plantsproduced by growing the seed of the inbred corn plant 3323, a sample ofsaid seed having been deposited under ATCC Accession No. ______.
 8. Acorn plant capable of expressing all the physiological and morphologicalcharacteristics of the inbred corn plant 3323, a sample of the seed ofsaid inbred corn plant 3323 having been deposited under ATCC AccessionNo. ______.
 9. The corn plant of claim 8, further comprising a factorconferring male sterility.
 10. A tissue culture of regenerable cells ofinbred corn plant 3323, wherein the tissue regenerates plants capable ofexpressing all the physiological and morphological characteristics ofthe inbred corn plant 3323, a sample of the seed of said inbred cornplant 3323 having been deposited under ATCC Accession No. ______. 11.The tissue culture of claim 10, wherein the regenerable cells comprisecells derived from embryos, immature embryos, meristematic cells,immature tassels, microspores, pollen, leaves, anthers, roots, roottips, silk, flowers, kernels, ears, cobs, husks, or stalks.
 12. Thetissue culture of claim 11, wherein the regenerable cells compriseprotoplasts or callus.
 13. A corn plant regenerated from the tissueculture of claim 10, wherein said corn plant is capable of expressingall of the physiological and morphological characteristics of the inbredcorn plant designated 3323, a sample of the seed of said inbred cornplant designated 3323 having been deposited under ATCC Accession No.______.
 14. An inbred corn plant cell of the corn plant of claim 8, saidcell comprising: (a) an SSR genetic marker profile in accordance withthe profile shown in Table 6; or (b) a genetic isozyme typing profile inaccordance with the profile shown in Table
 7. 15. A corn seed comprisingthe inbred corn plant cell of claim
 14. 16. A tissue culture comprisingthe inbred corn plant cell of claim
 14. 17. The inbred corn plant ofclaim 8, comprising: (a) an SSR genetic marker profile in accordancewith the profile shown in Table 6; or (b) a genetic isozyme typingprofile in accordance with the profile shown in Table
 7. 18. A processof producing corn seed, comprising crossing a first parent corn plantwith a second parent corn plant, wherein said first or second corn plantis the inbred corn plant 3323, a sample of the seed of said inbred cornplant 3323 having been deposited under ATCC Accession No. ______,wherein seed is allowed to form.
 19. The process of claim 18, furtherdefined as a process of producing hybrid corn seed, comprising crossinga first inbred corn plant with a second, distinct inbred corn plant,wherein said first or second inbred corn plant is the inbred corn plant3323, a sample of the seed of said inbred corn plant 3323 having beendeposited under ATCC Accession No. _______.
 20. The process of claim 19,wherein crossing comprises the steps of: (a) planting in pollinatingproximity seeds of said first and second inbred corn plants; (b)cultivating the seeds of said first and second inbred corn plants intoplants that bear flowers; (c) emasculating the male flowers of saidfirst or second inbred corn plant to produce an emasculated corn plant;(d) allowing cross-pollination to occur between said first and secondinbred corn plants; and (e) harvesting seeds produced on saidemasculated corn plant.
 21. The process of claim 20, further comprisinggrowing said harvested seed to produce a hybrid corn plant.
 22. Hybridcorn seed produced by the process of claim
 20. 23. A hybrid corn plantproduced by the process of claim
 21. 24. The hybrid corn plant of claim23, wherein the plant is a first generation (F₁) hybrid corn plant. 25.The corn plant of claim 4, further comprising a single locus conversion.26. The corn plant of claim 25, wherein the single locus was stablyinserted into a corn genome by transformation.
 27. The corn plant ofclaim 25, wherein the locus is selected from the group consisting of adominant allele and a recessive allele.
 28. The corn plant of claim 25,wherein the locus confers a trait selected from the group consisting ofherbicide resistance, insect resistance, resistance to bacterial,fungal, nematode or viral disease, yield enhancement, waxy starch,improved nutritional quality, enhanced yield stability, male sterilityand restoration of male fertility.